Materials and Methods Useful to Induce Cell Death via Methuosis

ABSTRACT

The present invention provides materials and methods to induce cell death by methuosis, a non-apoptotic cell death mechanism. Small molecules herein are useful for treating cell proliferation disorders or anomalies, particularly, but not exclusively, cancer. Methods related to the research and pharmaceutical use of the small molecules are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the authority of the Patent CooperationTreaty and claims priority to U.S. Provisional Application Ser. No.61/446,354 filed under 35 U.S.C. §111(b) on Feb. 24, 2011; thedisclosures of all priority applications are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Number R01CA115495 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention pertains to the field of biology, chemistry and medicine.The invention specifically pertains to materials and methods to inducemethuosis, a form of non-apoptotic cell death.

BACKGROUND OF THE INVENTION

Several different forms of non-apoptotic death have, based on specificmorphological or molecular criteria. These include death associated withaccumulation of autophagosomes, as well as several types ofcaspase-independent cell death that can represent specialized forms ofnecrosis; e.g., oncosis, necroptosis and paraptosis. A unique type ofnon-apoptotic cell death can be induced in glioblastoma and gastriccarcinoma cells by constitutive stimulation of Ras signaling pathways.This unique form of cell death is distinct from other kinds ofnon-apoptotic death noted above. It involves stimulation ofmacropinocytosis (cell drinking), combined with defects inclathrin-independent endocytic vesicle trafficking, ultimately resultingin accumulation of large vacuoles that disrupt cellular membraneintegrity. The unique form of cell death is termed ‘methuosis’, from theGreek methuo, to drink to intoxication. Mechanistically, the effects ofRas overexpression are related to activation of Rac1 and inactivation ofArf6, two GTPases implicated in macropinocytosis and endosome recycling,respectively.

Cancer cells typically harbor mutations in tumor suppressor genes thatcontrol programmed cell death, rendering them relatively insensitive toapoptosis. Moreover, many tumors that initially respond to treatmentwith chemotherapeutic drugs eventually develop multi-drug resistance dueto increases in drug efflux mechanisms or DNA repair capacity. Thesechallenges have stimulated interest in identifying alternative celldeath pathways that can be used to kill tumor cells that have ceased torespond to drugs that depend on induction of apoptotic mechanisms.

SUMMARY OF THE INVENTION

Without wishing to be bound by a particular theory, embodiments ofcompounds, compositions and methods of the invention can act viamethuosis to be effective in the treatment of cancer cells.

Disclosed herein is a chalcone-related compound that can rapidly inducecell death with the hallmarks of methuosis in bothtemozolomide-resistant and non-resistant glioblastoma cells, raising thepossibility that it can serve as a prototype for a new class oftherapeutic agents that can be used to treat tumors that are resistantto conventional drugs.

In one embodiment there are provided compounds having the structure ofFormula I:

wherein X and Y are independently absent or halogen; oxygen; azide;nitrogen; (CO)O; O(CO); O(CO)O; (CO)N; NH(CO); NH(CO)N; NH(CO)O; orO(CO)N, wherein if X or Y is halogen or azide, then R is absent, andwherein X or Y is nitrogen, (CO)N, or O(CO)N, or NH(CO)N, then two Rgroups are present;

wherein R and R₁ are independently hydrogen; alkyl, alkenyl, alkynyl,aryl or aralkyl;

wherein Ar is aryl;

wherein the dashed line is an optional double bond;

wherein the wavy line indicates that when said double bond is present,the resulting stereochemistry can be either cis or trans;

wherein when XR is hydrogen and Y is absent, then R is not hydrogen ormethyl; and

wherein when XR is halide and Y is absent, then R is not hydrogen; or

pharmaceutically acceptable salts, hydrates, and optical isomersthereof.

Also provided herein are compounds of Formula I, wherein the wavy lineindicates that a trans double bond is present; wherein XR is methoxybound at the 5 position, YR is methyl, and R1 is hydrogen; wherein Ar is3,4,5-trimethoxyyphenyl; and wherein Ar is 4-pyridyl.

In yet another embodiment, there are provided herein compounds havingthe structure of Formula II:

wherein X and Y are independently absent or halogen; oxygen; azide;nitrogen; (CO)O; O(CO); O(CO)O; (CO)N; NH(CO); NH(CO)N; NH(CO)O; orO(CO)N, wherein if X or Y is halogen or azide, then R is absent, andwherein X or Y is nitrogen, (CO)N, or O(CO)N, or NH(CO)N, then two Rgroups are present;

wherein R and R₁ are independently hydrogen; alkyl, alkenyl, alkynyl,aryl or aralkyl;

wherein Ar is aryl;

wherein the dashed line is an optional double bond;

wherein the wavy line indicates that when said double bond is present,the resulting stereochemistry can be either cis or trans; or

pharmaceutically acceptable salts, hydrates, and optical isomersthereof.

Also provided herein are compounds of Formula II wherein the wavy lineindicates that a trans double bond is present; wherein XR is methoxybound at the 5 position, YR is methyl, and R1 is hydrogen; wherein Ar is3,4,5-trimethoxyyphenyl; and wherein Ar is 4-pyridyl.

In yet another embodiment, there are provided methods of inducing celldeath in at least one cell, comprising introducing a compound of claim 1or claim 6 or both, to at least one cell and inducing cell death.Wherein the at least one cell is a mammalian cell; wherein the at leastone cell is apoptosis-resistant; wherein the at least one cell is acancer cell; wherein the at least one cell is in vitro; wherein the atleast one cell is an animal research model for cancer; wherein theanimal research model is for apoptosis-resistant cancer; and wherein theat least one cell is at least one human cell.

In another embodiment, there are provided methods of inducing cell deathin a mammal in need of such induction administering to a subject apharmacologically effective amount of a compound of Formula I or FormulaII, or both. Wherein the mammal is selected from the group consistingof: mouse; rat; guinea pig; rabbit; cat; dog; monkey; goat; cow; horse;and human; and wherein the mammal is a human.

In yet another embodiment, there are provided methods of amelioratingthe effects of cancer in a mammal in need of such amelioration,comprising administering to a subject a pharmacologically effectiveamount of a compound of Formula I or Formula II, or both. Also providedare methods wherein the cancer is selected from the group consisting of:brain, bladder, lung, liver, pancreas, bone, colon, stomach, breast,prostate, ovary, central nervous system, or skin cancer; wherein themammal is selected from the group consisting of: mouse; rat; guinea pig;rabbit; cat; dog; monkey; goat; cow; horse; and human; wherein themammal is a human.

Also provided are methods wherein the method further comprisesadministering a second compound, adjuvant or additional therapeutic tothe mammal. Also provided are methods which further comprise physicalremoval of glioblastoma cells via a method selected from the groupconsisting of: surgery; aspiration; dissection; ablation; andelectromagnetic fluctuations.

In yet another embodiment, there are provided compositions of mattercomprising a compound of Formula I or Formula II, or both, and a cancertherapeutic. Also provided are compositions wherein the cancertherapeutic is selected from the group consisting of chemotherapeuticdrug; toxin; immunological response modifier; enzyme; gamma radiation,and radioisotope.

In yet another embodiment, there are provided methods of amelioratingthe effects of cellular proliferation disorder in a mammal in need ofsuch amelioration, comprising administering to a subject apharmacologically effective amount of a compound of Formula I or FormulaII, or both.

In yet another embodiment, there are provided compounds selected fromthe group consisting of:trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(MOMIPP); 2-Methylindole-3-carboxaldehyde (compound 2a);trans-3-(2-Methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 2); trans-3-(1H-Indol-3-yl)-1-phenyl-2-propen-1-one (compound9); trans-3-(1H-Indol-3-yl)-1-(2-pyridinyl)-2-propen-1-one (compound10); trans-3-(1H-Indol-3-yl)-1-(3-pyridinyl)-2-propen-1-one (compound11); trans-3-(1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (compound12); trans-3-(5-Methoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 13); trans-3-(5-Phenylmethoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (compound 14);trans-3-(5-Hydroxy-1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 15);trans-3-(5-Methoxy-1H-indol-3-yl)-1-(3-pyridinyl)-2-propen-1-one(compound 16);trans-3-(5-Methoxy-1H-indol-3-yl)-1-(pyrazine)-2-propen-1-one (compound17); 5-Methoxy-2-methyl-1H-indole-3-carboxaldehyde (compound 18);trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 19);trans-3-(5-Methoxy-1-methyl-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 20);trans-3-(5-Hydroxy-1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 21); 2-Methyl-1H-indol-5-ol (compound 22);5-(4-Methylbenzoate)methoxy-2-methyl-1H-indole (compound 23);5-(4-Methylbenzoate)methoxy-2-methyl-1H-indole-3-carboxaldehyde(compound 24); 5-(4-Benzoate)methoxy-2-methyl-1H-indole-3-carboxaldehyde(compound 25);trans-3-[5-((4-Methylbenzoate)methoxy)-1H-Indol-3-yl)]-1-(4-pyridinyl)-2-propen-1-one(compound 26);trans-3-[5-((4-Carboxyphenyl)-methoxy)-1H-Indol-3-yl)]-1-(4-pyridinyl)-2-propen-1-one(compound 27); 2-Methyl-5-benzoyl-indole-3-carboxaldehyde (compound 28);2-Methyl-6-benzoyl-indole-3-carboxaldehyde (compound 29);2-Methyl-5-benzoyl-indole-3-carboxaldehyde (compound 28);2-Methyl-6-benzoyl-indole-3-carboxaldehyde (compound 29);trans-3-(5-Benzoyl-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 30);trans-3-(6-Benzoyl-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 31); 2-Methyl-5-nitro-1H-indole (compound 32);2-Methyl-5-amino-1H-indole (compound 33); 2-Methyl-5-azido-1H-indole(compound 34); 2-Methyl-3-carboxaldehyde-5-azido-1H-indole (compound35);trans-3-(5-Azido-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 36); 2-Methyl-5-methoxy-6-nitro-1H-indole (compound 37);2-Methyl-5-methoxy-6-amino-1H-indole (compound 38);2-Methyl-5-methoxy-6-azido-1H-indole (compound 39);2-Methyl-3-carboxaldehyde-5-methoxy-6-azido-1H-indole (compound 40); andtrans-3-(6-Azido-5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 41), or pharmaceutically acceptable salts, hydrates, andoptical isomers thereof.

Also provided are compounds selected from FIG. 48, or pharmaceuticallyacceptable salts, hydrates, and optical isomers thereof.

In particular, compound #2 (MIPP) of FIG. 48 is provided, orpharmaceutically acceptable salts, hydrates, and optical isomersthereof.

In particular, compound #2 (MIPP) of FIG. 48 is provided, orpharmaceutically acceptable salts, hydrates, and optical isomersthereof.

In particular, compound #12 of FIG. 48 is provided, or pharmaceuticallyacceptable salts, hydrates, and optical isomers thereof.

In particular, compound #13 of FIG. 48 is provided, or pharmaceuticallyacceptable salts, hydrates, and optical isomers thereof.

In particular, compound #14 of FIG. 48 is provided, or pharmaceuticallyacceptable salts, hydrates, and optical isomers thereof.

In particular, compound #19 (MOMIPP) of FIG. 48 is provided, orpharmaceutically acceptable salts, hydrates, and optical isomersthereof.

Various aspects of this invention will become apparent to those skilledin the art from the following detailed description of the preferredembodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file can contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIG. 1. Compounds, I and II, induce extreme cytoplasmic vacuolization inU251 glioblastoma cells.

FIGS. 2-5. Vacuoles induced by MIPP are derived from macropinosomes thatundergo progressive fusion events and accumulate at a pre-lysosomalstage. FIG. 2. Time-lapse phase-contrast microscopy of U251 cellstreated with MIPP. The small two-headed arrows point to vesicles thathave fused in the subsequent frame. FIG. 3. The same field of cells isdepicted in the matching phase-contrast and fluorescent images. In thetop left panel, the arrows indicate some of the specific vacuoles thathave incorporated the Lucifer yellow. FIG. 4. Phase-contrast images ofvacuoles induced by MIPP, showing that Filipin blocks the induction ofvacuoles. FIG. 5. Phase-contrast images of Bafilomycin A1 (Baf-A)blocking the induction of vacuoles by MIPP.

FIG. 6. Vacuoles induced by MIPP acquire characteristics of lateendosomes, but remain distinct from autophagosomes.

FIGS. 7-11. MIPP affects the activation states of Rab5 and Rab7, but notRac1 or Arf6. In separate experiments U251 cells were treated with 10 μMMIPP for the indicated periods of time and then harvested for pull downassays to measure the relative amounts of active Rac1 (FIG. 7), Arf6(FIG. 8), Rab5 (FIG. 9) or Rab7 (FIG. 10). As an additional control, thestudies of Rab7 were conducted with cells treated with the inactivecompound III instead of MIPP (FIG. 11).

FIGS. 12-17. MIPP induced vacuolation leads to non-apoptotic cell deathin glioblastoma cells. FIG. 12. MTT assay of U251 cells treated overtime with the indicated compounds (refer to FIG. 1 for structures) at aconcentration of 10 μM. The arrows point to vacuolated cells that haverounded and detached from the surface of the dish. FIG. 13. ATP levelsdecline in cells treated with MIPP. FIG. 14. The arrows point to thevacuolated cells that have rounded and detached from the surface of thedish in cultures treated with MIPP. FIG. 15. U251 cells were treatedwith 10 μM MIPP or an equivalent volume of DMSO (control) for 2 days andcolony forming assays were performed. FIG. 16. U251 cells were examinedby electron microscopy after two days of treatment with 10 μM MIPP. Thearrows point to regions of plasma membrane discontinuity indicative ofcell rupture. FIG. 17. Inhibition of caspase activity does not preventMIPP-induced cell death.

FIGS. 18-21. MIPP has similar effects on cell morphology and viabilityin temozolomide-resistant (U251-TR) and parental (U251) glioblastomacells. FIG. 18. Cells were treated with the indicated concentrations oftemozolomide for 48 h. FIG. 19. Phase-contrast images of cells aftertreatment for 48 h with 10 μM MIPP or an equivalent volume of DMSO(control). FIG. 20. MTT assays were performed after treatment for 48 hwith the indicated concentrations of MIPP or an equivalent volume ofDMSO. FIG. 21. U251-TR cells were treated with 10 μM MIPP or anequivalent volume of DMSO (control) for 2 days and colony forming assayswere performed.

FIG. 22. U251 cells were transfected with expression vectors encodingEGFP-Rab5 or EGFP-Rab7.

FIGS. 23-25. The Rac inhibitor, EHT 1864, does not block the inductionof vacuoles by MIPP. FIG. 23. U251 cells were incubated with MIPP for 24h in the presence or absence of 25 μM EHT 1864. FIG. 24. In a separateexperiment, U251 cells were incubated with or without EHT 1864 followingnucleofection with a vector encoding a constitutively active H-Ras(G12V). FIG. 25. Phase-contrast images were taken 24 h after addition ofthe Rac inhibitor. The scale bars are 10 microns.

FIGS. 26-28. MIPP induces vacuoles and inhibits growth and viability inmultiple human cell lines. FIG. 26. Phase-contrast images of cells wereacquired after two days of treatment with 10 μM MIPP. FIG. 27. MTTassays were performed on cells treated for the indicated number of dayswith 10 μM MIPP or an equivalent volume of DMSO. FIG. 28. Colony-formingassays for the transformed cell lines.

FIG. 29. Compounds related to compound 2 (MIPP) identified from databasesearches and purchased from commercial suppliers.

FIG. 30. Reaction scheme for condensation of various acetyl-pyridines oracetophenone with indole-3-carboxaldehyde to obtain compounds 9-12.

FIG. 31. Reaction scheme for demethylation of compound 13 to produce the5-OH derivative, compound 15.

FIG. 32. Reaction scheme for synthesis of compound 19 viaVilsmeier-Haack formylation, followed by coupling with acetyl-pyridine.

FIG. 33. Reaction scheme for methylation of the indole nitrogen ofcompound 13 to yield compound 20.

FIG. 34. Reaction scheme for generating compound 21 by demethylation ofcompound 19.

FIG. 35. Reaction scheme for generating polar analogs of compound 19 byfunctionalizing the indole ring before introduction of the 4-pyridinemoiety.

FIG. 36. Structure-activity relationships of a directed library ofcompounds related to compound 2 (MIPP). The R₁, R₂, R₃ and Ar groupsrefer to positions designated in the structures depicted in FIGS. 30-35.

FIG. 37A-37D. Dose-response comparison of the effects of MOMIPP versusMIPP, 5-azido MIPP and 6-azido-MIPP on the morphology and viability ofU251 glioblastoma cells.

FIG. 38. Effects of MOMIPP versus MIPP on cell proliferation andviability determined by cell number.

FIG. 39. Comparison of the effects of MIPP versus MOMIPP on themorphology of U251 cells

FIG. 40. Comparison of the effects of MOMIPP versus MIPP oncolony-forming ability of U251 glioblastoma cells after long term (2days) or short term (4 hours) treatment.

FIGS. 41A-41H. MOMIPP effectively inhibits the viability ofdrug-resistant GBM cells and breast cancer cells.

FIG. 42. Effects of MOMIPP on the morphology and viability of multiplehuman glioblastoma cell lines, including CD133⁺ GBM stem cells.

FIG. 43. Effects of MOMIPP on colony-forming ability of multiple humanGBM cell lines.

FIG. 44. MOMIPP causes vacuolization and inhibits colony formation incultured GBM stem cells isolated directly from a human tumor.

FIG. 45. Effects of MOMIPP on the morphology of wild-type MCF-7 anddoxorubicin-resistant (MCF-7_(DOX)) mammary carcinoma cells.

FIG. 46. Effects of MOMIPP on the short-term viability of wild-typeMCF-7 and doxorubicin-resistant (MCF-7_(DOX)) mammary carcinoma cells.

FIG. 47. Effects of MOMIPP on the colony-forming ability of wild-typeMCF-7 and doxorubicin-resistant (MCF-7_(DOX)) mammary carcinoma cells.

FIG. 48. Table of summary of SAR studies performed on MIPP (compound 2)and related compounds.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a specific chalcone-like molecule,3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MIPP) that isshown to induce cell death with the hallmarks of methuosis. MIPP causesrapid accumulation of vacuoles that can be labeled with extracellularfluid phase tracers. Vacuolization can be blocked by an inhibitor of thevacuolar-type Fr-ATPase, bafilomycin A1, and by thecholesterol-interacting compound, filipin, consistent with the endosomalorigin of the vacuoles. Although the vacuoles acquire somecharacteristics of late endosomes, they remain distinct from lysosomaland autophagosomal compartments, showing a block at the lateendosome/lysosome boundary.

MIPP targets steps in the endosomal trafficking pathway mediated by Rab5and Rab7, as shown by changes in the activation states of these GTPases.These effects are specific, as other GTPases (Rac1, Arf6) are unaffectedby the compound. Cells treated with MIPP lose viability within 2-3 days,but their nuclei show no apoptotic changes. Inhibition of caspaseactivity does not protect the cells, consistent with a non-apoptoticdeath mechanism. A U251 glioblastoma line selected for temozolomideresistance showed sensitivity to MIPP-induced methuosis that wascomparable to the parental cell line. MIPP can serve as a prototype fornew drugs that can be used to induce non-apoptotic death in cancers thathave become refractory to agents that work through DNA damage andapoptotic mechanisms.

Glioblastomas are highly aggressive brain tumors that almost alwaysrecur after surgery. Treating these tumors is extremely challengingbecause the residual cells are highly invasive and they typically harborgenetic mutations that decrease their sensitivity to apoptosis inducedby radiation or DNA alkylating agents. Thus, the discovery of cell deathmechanisms that do not depend on activation of the classicalmitochondrial or death receptor-mediated apoptotic pathways presents newopportunities to treat these devastating tumors. A non-conventional formof cell death termed methuosis can be triggered by ectopic expression ofconstitutively activated Ras in glioblastoma and other cancer celllines.

In this disclosure, a small molecule termed MIPP has been identified,which induces the hallmark cytopathological features of methuosis.Shortly after being exposed to MIPP, glioblastoma cells exhibit influxof multiple macropinocytotic vesicles. In the time-lapse studies, thelatter can be seen undergoing fusion events to form larger vacuoles. Asthe vacuoles accumulate, they rapidly acquire some characteristics oflate endosomes (Rab7, LAMP1), but they do not merge with lysosomalcompartments. Ultimately, the displacement of much of the cytoplasmicspace by the vacuoles is accompanied by a decline in metabolic activityand a necrosis-like rupture of the cell. Consistent with a non-apoptoticmechanism, these changes cannot be prevented by caspase inhibitors, andnuclear chromatin condensation typical of apoptosis is not observed.Further, in this disclosure it is shown that glioblastoma cells selectedfor resistance to the front-line chemotherapeutic drug, temozolomide,were susceptible to MIPP-induced methuosis by comparingdoxorubicin-resistant versus non-resistant MCF-7 breast cancer cellstreated with a MIPP analog. It is disclosed herein that MIPP can serveas a prototype for development of drugs that can be used to triggerdeath by methuosis in drug-resistant cancers.

Further disclosed herein is the ability of compounds 1 (BIPP) and 2(MIPP) to induce methuosis. A similarity search of chemical databaseswas performed using the Chembridge Hit2Lead search engine, whichprovided compounds 3-8, as shown in FIG. 29, for analysis of whethercertain similarly-structured compounds can induce methuosis. Whentesting these compounds in U251 glioblastoma cells at 10 μM, allcompounds were completely inactive in inducing methuosis and limitingcell growth.

In certain embodiments, based on the data of compounds 3-8, the3-indole-1-pyridinyl-2-propen-1-one scaffold was used to inducemethuosis. Compounds 3-8 all possess this core, yet were inactive. Theinactivity of compound 8 is most revealing when compared to activecompound 1, indicating that addition of a para-methoxy in place of thepyridine nitrogen rendered the compound inactive.

Analogs based on the α,β-unsaturated ketone core were designed fromClaisen-Schmidt condensations between indole-3-carboxaldehydes andaromatic ketones. Such aldol condensations are known to proceed wellemploying secondary amines as catalysts. Initially, the orientation ofthe pyridine nitrogen was investigated. Condensation of variousacetyl-pyridines or acetophenone with indole-3-carboxaldehyde (FIG. 30)yielded compounds 9-12 (FIGS. 36 and 48). The activities of thesecompounds were compared to compound 2 (i.e., MIPP), at a concentrationof 10 μM using three criteria: 1) morphological vacuolization of livecells assessed by phase contrast microscopy at 24 h and 48 h, 2) cellviability, assessed at a 48 h end point by MTT assay, with freshcompound added after the first 24 h, and 3) colony forming assays (twoweek end-point) performed on cells exposed to the compounds for 48 h.The results of this analysis (FIGS. 36 and 48) showed that apara-nitrogen orientation of the pyridine ring is a key feature requiredfor activity. The meta and ortho analogs 10 & 11, as well as theacetophenone analog 9 were all relatively ineffective at inducingmethuosis compared with MIPP. In contrast, removal of the 2-methyl fromthe indole ring of MIPP reduced but did not eliminate activity.

Next, the consequences of functionalizing the 5-position of the indolering with the suitable 5-methoxy and 5-benzyloxyindole-3-carboxaldehydes, compounds 13 and 14 was explored. The5-methoxy compound 13 was demethylated with BBr₃, affording the 5-OHcompound 15 (FIG. 31). As shown in FIG. 36 and FIG. 48, the activitiesof compounds 13 and 14 were similar to compound 2 (MIPP) when comparedby colony-forming assay and cell morphology, although they were not aseffective in the short-term viability assay. In contrast, addition ofthe OH to the 5-position greatly reduced activity of the compound in allof the assays. To verify that the location of the pyridine nitrogen wasstill crucial in the 5-substituted compounds, analogs 16 and 17 weregenerated. Loss of activity confirmed the necessity of the para-nitrogen(FIGS. 36 and 48).

After comparing compounds 13 and 14 to MIPP, which showed thatmodifications at the 5- and 2-positions of the indole ring both affectthe activity of these compounds, a 2-methyl-5-methoxy analog wasgenerated from available 2-methyl-5-methoxy-indole. Compound 19 wassynthesized by a Vilsmeier-Haack formylation, followed by coupling with4-acetyl-pyridine (FIG. 32). The activity of this compound exceeded thatof compound 2 in both the MTT viability assay and the colony formationassay (FIGS. 36 and 48).

The addition of the methyl at the indole's 2 position in compound 19gave added potency versus compound 13, possessing only the 5-methoxy.Methylation of the indole-nitrogen of compound 13 with NaH/CH₃I in DMF(FIG. 33) to yield 20, produced a compound that induced some cytoplasmicvacuolization but had modest effects on cell viability. Thus, aftercomparing compounds 11, 19 and 20, it is shown that optimal activity isachieved when the 1 and 2 position of the indole are occupied by H andmethyl, respectively. The 5-OH compound 21, made by BBr₃ demethylationof compound 19 (FIG. 34), showed a marked reduction in activity comparedto the 5-methoxy compound 19, consistent with the detrimental effect ofthe 5-OH in compound 15.

Further disclosed herein are the effects of modifying the 5-position ofthe indole with a group that would add polarity at physiological pH. Ananalog of compound 14 was designed, that added charge and also includeda methyl at the indole's 2 position. No appreciable amount of productwas produced by direct alkylation of compound 21. Multiple bases werescreened with varying pKa's, from K₂CO₃ and Cs₂CO₃, as well astriethylamine, DBU, TMG and NaH. In all reactions, alkylation at thepyridine nitrogen was observed.

Stronger bases such as TMG and NaH, even using one equivalent, producedappreciable indole-N-alkylation as well as pyridine and indole-OHalkylation. The yield of singly alkylated product at the 5-indoleposition was negligible. Thus, a new route was designed byfunctionalizing the indole before introduction of the pyridine moiety(FIG. 35). Available 2-methyl-5-methoxy-indole was demethylated withBBr₃ providing compound 22, followed by alkylation with4-methyl-(bromomethyl)-benzoate under phase-transfer conditions toafford the singly O-alkylated product compound 23. After formylationwith POCl₃/DMF, compounds 24 and 25 were selectively made. Workup inmild base (NaHCO₃) produced the ester compound 24, while workup in 5NNaOH gave acid compound 25. Condensations with 4-acetyl-pyridinesprovided the corresponding targets compounds 26 and 27. However, neithercompound 26 nor compound 27 induced methuosis (FIGS. 36 and 48).

Superior biological activity of MOMIPP versus MIPP was confirmed instudies with U251 glioblastoma cells, using MTT viability assays, cellgrowth assays, morphological assessment, and colony forming assays tocompare the two compounds. FIGS. 37A-37D show the dose-response curvesfor the effects of the two drugs on cell viability. Each compound wasadded at the mentioned concentration for two days, with medium andcompounds replenished after the first day. The relative IC₅₀ for MOMIPPwas 1.9 μM, versus 4.8 μM for MIPP.

To obtain a measure of the comparative duration of activity for eachcompound, their effects on cell growth and survival were assessed bycounting the number of cells in parallel cultures treated for threeconsecutive days with 2.5 μM, 5 μM, or 10 μM compound (FIG. 38). Unlikethe viability studies, in these experiments the compounds were added atthe beginning of the study and were not replenished for the duration.Under these conditions, MOMIPP was shown to be more effective than MIPPin reducing cell growth and viability. The reduction of cell number inthe cultures treated with MOMIPP coincided with massive earlyvacuolization of the cells and loss of nonviable cells from thesubstratum (FIG. 39).

In contrast, the cells treated with MIPP initially underwentvacuolization on days 1 and 2, but tended to recover, especially at thelower concentrations of the compound (FIG. 39). These studies show thata single application of MOMIPP has a more sustained effect than MIPP oncell morphology and cell viability. The difference between the twocompounds was underscored when colony-forming assays were used toevaluate proliferative capacity and long-term cell viability (FIG. 40).MOMIPP was shown to be more effective than MIPP in reducing colonyformation when cells were treated for 2 days (FIG. 40).

In yet another embodiment, it is shown that when treatment was shortenedto just 4 h, MOMIPP was still more effective than MIPP, but higherconcentrations of both compounds were required to reduce colonyformation (FIG. 40).

The methuosis-inducing compounds are effective for targetingtemozolomide (TMZ)-resistant glioblastoma. As shown in FIGS. 41A and41B, both MIPP and MOMIPP induced methuosis and reduced cell viabilityin the TMZ-resistant cells, but the efficacy of MOMIPP was shown to besuperior to MIPP.

The ability of MOMIPP to kill drug-resistant tumor cells by methuosisextends beyond glioblastoma. For example, comparable levels ofMOMIPP-induced methuosis was observed in parental anddoxorubicin-resistant MCF-7 breast cancer cells, as shown in FIGS.41C-41D, 45 and 46.

The methuosis-inducing activity of MOMIPP was further shown to extend tomultiple human GBM cell lines with different genetic backgrounds, asshown in FIGS. 42 and 43.

The methuosis-inducing activity of MOMIPP was also shown to extend tohuman CD133⁺ GBM stem cell populations, as shown in FIGS. 42 and 44.

The mechanism by which MIPP causes cellular vacuolization and death caninvolve three steps; 1) a transient increase in macropinocytoticactivity, 2) rapid homotypic fusion of the macropinosome-derivedvesicles to form vacuoles that acquire late endosomal characteristics,and 3) failure of the resulting vacuoles to recycle or fuse withlysosomes, leading to their progressive accumulation. Similar steps canbe involved in the death mechanism triggered by MOMIPP, as shown inFIGS. 37-44.

Ras-induced methuosis depends on activation of Rac1 and reciprocalinactivation of Arf6, leading to hyperstimulation of macropinocytosisand defects in macropinosome recycling and downstream trafficking.However, the measurements of the activation states of Rac1 and Arf6 didnot reveal any changes in MIPP treated cells. On the other hand, MIPP isshown to have profound effects on the activities of Rab5 and Rab7,decreasing the amount of active Rab5 and increasing the amount of Rab7.This shows that the compound operates directly on the machinery thatregulates the trafficking and fusion of endosomal vesicles.

Rab5 controls receptor-mediated (clathrin-dependent) endosome formation,homotypic fusion, and delivery of early endosomes to thesorting/recycling compartment. The latter serves as a “way station” fromwhich early endosomes can either recycle to the cell surface or progressto late endosomes in a multi-step process involving Rab5-Rab7conversion.

While not wishing to be bound by theory, it is further disclosed hereinthat the main consequence of the MIPP-induced decrease in active Rab5can occur after the initial formation of macropinosomes. That is, thenascent vesicles formed during the initial burst of macropinocytoticactivity fail to fuse with EEA1-positive early endosomes and thus do notenter the sorting/recycling compartment. Instead they immediatelyacquire the capability to recruit LAMP1 and Rab7. The resulting vesiclesthen undergo abnormal homotypic fusions to form progressively largervacuoles in a manner that requires activity of the H⁺-ATPase.

Despite their ability to undergo homotypic fusion, the apparentinability of the Rab7-positive vacuoles to merge with lysosomes showingthat they can lack some of the key protein components of complexes thatare required for heterotypic tethering and fusion of late endosomes withlysosomes and autophagosomes (e.g., HOPS, trans-SNARE). This can explainthe progressive accumulation of the vacuoles and the overall increase inthe amount of active Rab7, as GAP-mediated GTP-hydrolysis that wouldnormally coincide with endosome-lysosome fusion fails to occur.

Based on the examination of several cancer cell lines, it is shown thatthe ability of MIPP to induce methuosis extends beyond glioblastoma. Itis further disclosed herein that MIPP can also trigger vacuolization anda modest reduction of cell proliferation/viability in normalproliferating cells, which can lead to delivery of such compounds totumors.

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to texts, journal referencesand contexts known to those skilled in the art.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably. The expression “of any ofclaims XX-YY” (wherein XX and YY refer to claim numbers) is intended toprovide a multiple dependent claim in the alternative form, and in someembodiments is interchangeable with the expression “as in any one ofclaims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers and enantiomers of the group members, are disclosed separately.When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.It is intended that any one or more members of any Markush group orlisting provided in the specification can be excluded from the inventionif desired. When a compound is described herein such that a particularisomer or enantiomer of the compound is not specified, for example, in aformula or in a chemical name, that description is intended to includeeach isomers and enantiomer of the compound described individual or inany combination. Additionally, unless otherwise specified, all isotopicvariants of compounds disclosed herein are intended to be encompassed bythe disclosure. For example, it will be understood that any one or morehydrogens in a molecule disclosed can be replaced with deuterium ortritium. Isotopic variants of a molecule are generally useful asstandards in assays for the molecule and in chemical and biologicalresearch related to the molecule or its use. Specific names of compoundsare intended to be exemplary, as it is known that one of ordinary skillin the art can name the same compounds differently.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing components that aredescribed in the publications that might be used in connection with thepresently described invention.

The following abbreviations are applicable. Baf-A, bafilomycin A1; DMEM,Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; FBS, fetalbovine serum; GAP, GTPase activating protein; LAMP1,lysosomal-associated membrane protein 1; LC3, microtubule-associatedprotein light chain 3; MIPP, 3-(2-Methyl-1Hindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one; MOMIPP,[trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one];MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SAR,structure-activity relationships; and TMZ, temozolomide.

The definitions are provided to clarify their specific use in thecontext of the invention.

Thus, as used herein, the term “alkyl” includes straight, branched andcyclic alkyl groups, having up to 10 carbon atoms. An analogousconvention applies to other generic terms such as “alkenyl”, “alkynyl”and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”,“alkynyl” and the like encompass both substituted and unsubstitutedgroups. In certain embodiments, as used herein, “lower alkyl” is used toindicate those alkyl groups (cyclic, acyclic, substituted,unsubstituted, branched or unbranched) having 1-6 carbon atoms.

Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,—CH₂-cyclopropyl, allyl, n-butyl, secbutyl, isobutyl, tert-butyl,cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl,tert-pentyl, cyclopentyl, —CH₂-cyclopentyl-n, hexyl, sec-hexyl,cyclohexyl, —CH₂cyclohexyl moieties and the like, which again, can bearone or more substituents. Illustrative alkynyl groups include, but arenot limited to, for example propargyl.

“Aryl” refers to an unsaturated aromatic or heteroaromatic carbocyclicgroup of from 1 to 15 carbon atoms having a single ring (e.g. phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Preferred arylsinclude substituted aromatic C6-12 carbocycle; unsubstituted aromaticC1-10 heterocycle; substituted aromatic C1-10 heterocycle; wherein whensubstituted, the substitution is —XR.

Aralkyl refers to an alkyl herein connected to an aryl herein.

Unless otherwise constrained by the definition for the aryl substituent,such aryl groups can optionally be substituted with from 1 to 3substituents selected from the group consisting of hydroxy, acyl, alkyl,alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted alkynyl, amino, aminoacyl, aminocarboxyesters, alkaryl, aryl, aryloxy, carboxyl, carboxylalkyl, acylamino,cyano, halo, nitro, heteroaryl, heterocyclic, oxyacyl, oxyacylamino,thioalkoxy, substituted thioalkoxy, trihalomethyl, mono- anddi-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclicamino, and unsymmetric di-substituted amines having differentsubstituents selected from alkyl, substituted alkyl, aryl, heteroaryland heterocyclic, and the like.

“Halogen” refers to fluoro, chloro, bromo and iodo and preferably iseither chloro or bromo.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings, from 1 to 8carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfuror oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 3 substituents selected from the group consisting of hydroxy, acyl,alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted alkynyl, amino, aminoacyl, aminocarboxyesters, alkaryl, aryl, aryloxy, carboxyl, carboxylalkyl, aminoacyl,cyano, halo, nitro, heteroaryl, heterocyclic, oxyacyl, oxyacylamino,thioalkoxy, substituted thioalkoxy, trihalomethyl, mono- anddi-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclicamino, and unsynumetric di-substituted amines having differentsubstituents selected from alkyl, substituted alkyl, aryl, heteroaryland heterocyclic, and the like. Such heterocyclic groups can have asingle ring or multiple condensed rings. Preferred saturatedheterocyclics include morpholino, piperidinyl, and the like; andpreferred unsaturated heterocycles include pyridyl and the like.

Examples of heterocycles and heteroaryls include, but are not limitedto, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, indoline, phthalimide,1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene,thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino,piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

It will be appreciated by one of ordinary skill in the art thatasymmetric centers can exist in the compounds of the present invention.Thus, inventive compounds and pharmaceutical compositions thereof can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers. It is to beunderstood that the invention encompasses all possible isomers such asgeometric isomers, optical isomers, stereoisomers and tautomers based onan asymmetric carbon, which can occur in the structures of the inventivecompounds, and mixtures of such isomers and compositions comprisingthose compounds, and is not limited to the specific stereochemistryshown for the compounds disclosed in the present specification. It willbe further appreciated that the absolute stereochemistry of some of thecompounds recited in the Exemplification herein cannot have beendetermined, and that when a stereochemistry was assigned for thosecompounds it is meant to be tentative and to indicate that a set ofdiastereomers exists for those compounds and/or that a diastereomer wasisolated in pure form. Furthermore, it will be appreciated that certainof the compounds disclosed herein contain one or more double bonds andthese double bonds can be either Z or E, unless otherwise indicated. Incertain embodiments, the compounds of the invention are enantiopurecompounds. In certain other embodiments, mixtures of stereoisomers ordiastereomers are provided.

Additionally, the present invention provides pharmaceutically acceptablederivatives of the inventive compounds, and methods of treating asubject using these compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents. The phrase, “pharmaceutically acceptable derivative”, as usedherein, denotes any pharmaceutically acceptable salt, ester, or salt ofsuch ester, of such compound, or any other adduct or derivative which,upon administration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof.

Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups. By the term “protecting group”, has used herein, itis meant that a particular functional moiety, e.g., O, S, or N, istemporarily blocked so that a reaction can be carried out selectively atanother reactive site in a multifunctional compound. In preferredembodiments, a protecting group reacts selectively in good yield to givea protected substrate that is stable to the projected reactions; theprotecting group must be selectively removed in good yield by readilyavailable, preferably nontoxic reagents that do not attack the otherfunctional groups; the protecting group forms an easily separablederivative (more preferably without the generation of new stereogeniccenters); and the protecting group has a minimum of additionalfunctionality to avoid further sites of reaction.

For example, oxygen, sulfur, nitrogen and carbon protecting groups canbe utilized. Certain exemplary oxygen protecting groups include, but arenot limited to methyl ethers, substituted methyl ethers (e.g., MOM(methoxymethyl ether), MTM (methylthiomethyl ether), BOM(benzyloxymethyl ether), PMBM (p-methoxybenzyloxymethyl ether), to namea few), substituted ethyl ethers, substituted benzyl ethers, silylethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS(triisopropylsilyl ether), TB DMS (t-butyldimethylsilyl ether),tribenzyl silyl ether, TBDPS (t-butyldiphenyl silyl ether), to name afew), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate,dichloroacetate, to name a few), carbonates, cyclic acetals and ketals.

Exemplary nitrogen protecting groups include, but are not limited to,carbamates (including methyl, ethyl and substituted ethyl carbamates(e.g., Troc), to name a few) amides, cyclic imide derivatives, N-Alkyland N-Aryl amines, inline derivatives, and enamine derivatives, to namea few. As will be appreciated by those of ordinary skill in the art, avariety of additional equivalent protecting groups can be utilized inaccordance with the present invention.

It will be appreciated that the compounds, as described herein, can besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure can be substituted with more thanone substituent selected from a specified group, the substituent can beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this invention, heteroatoms such as nitrogencan have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms.

Furthermore, this invention is not intended to be limited in any mannerby the permissible substituents of organic compounds. The term “stable”,as used herein, preferably refers to compounds which possess stabilitysufficient to allow manufacture and which maintain the integrity of thecompound for a sufficient period of time to be detected and preferablyfor a sufficient period of time to be useful for the purposes detailedherein.

As used herein, the term “patient” is intended to include livingorganisms in which certain conditions as described herein can occur.Examples include humans, monkeys, cows, sheep, goats, dogs, cats, mice,rats, and transgenic species thereof. In a preferred embodiment, thepatient is a primate. In an even more preferred embodiment, the primateis a human. Other examples of subjects include experimental animals suchas mice, rats, dogs, guinea pigs, cats, goats, sheep, pigs, and cows.The experimental animal can be an animal model for a disorder, e.g., atransgenic mouse with cancer.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the Maximum response obtained.

Dosing

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount can vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions can comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound can comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition can beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimenscan be desirable.

In other non-limiting examples, a dose can also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Identification of Methuosis-Inducing Molecules

The examples describe the particular assay systems used to identifymethuosis-inducing molecules as described herein. As shown in theExamples, and as would be readily appreciated by those of ordinary skillin the art, additional or alternative methuosis-inducing molecules canreadily be identified by applying the same screen to other chemicalcompounds.

For instance, extracts containing natural products are often used assources of test compounds for biological assays. Alternatively oradditionally, synthetic compounds can be utilized. As will beappreciated by those of ordinary skill in the art, the development ofcombinatorial chemistry and split-pool synthesis techniques have addedto the repertoire complex compound libraries, the products of laboratorysyntheses, as a source of small molecules to be screened for compoundswith biological activity. However, small molecules synthesized byparallel synthesis methods and by traditional methods (one-at-a-timesynthesis and modifications of these structures) can also be utilized inthe compositions and methods of the present invention, as can naturallyoccurring compounds, or other collections of compounds.

As will be realized by one of ordinary skill in the art, insplit-and-pool techniques, a mixture of related compounds can be made inthe same reaction vessel, thus substantially reducing the number ofcontainers required for the synthesis of very large libraries, such asthose containing as many as or more than one million library members. Asan example, a solid support bound scaffold can be divided into nvessels, where n represents the number of species of reagent A to bereacted with the support bound scaffold. After reaction, the contentsfrom n vessels are combined and then split into m vessels, where mrepresents the number of species of reagent B to be reacted with thesupport bound scaffold. This procedure is repeated until the desirednumbers of reagents are reacted with the scaffold structures to yield adesired library of compounds.

As mentioned above, the use of parallel synthesis methods are alsoapplicable. Parallel synthesis techniques traditionally involve theseparate assembly of products in their own reaction vessels. Forexample, a microtiter plate containing n rows and m columns of tinywells which are capable of holding a small volume of solvent in whichthe reaction can occur, can be utilized. Thus, n variants of reactanttype A can be reacted with m variants of reactant type B to obtain alibrary of n×m compounds.

Preparation of Methuosis-Inducing Molecules

Once identified, methuosis-inducing molecules of the present inventioncan be prepared by any available means. In some cases, themethuosis-inducing molecules can be compounds that occur naturally andtherefore can be prepared from a natural source using known purificationand isolation technologies. In other cases, the methuosis-inducingmolecules cannot be naturally-occurring, or cannot be readily isolatablefrom a natural source, and therefore can instead be prepared usingsynthetic techniques, as is known in the art. Any appropriate synthetictechniques can be employed, including those that utilize only chemicalreagents or those that utilize biological reagents such as syntheticenzymes. Alternatively or additionally, synthetic and isolationarytechniques can be combined in the preparation of inventivemethuosis-inducing molecules. The terms “isolated” or “substantiallypurified” as used interchangeably herein refer to vacuolin compounds ina non-naturally occurring state. The compounds can be substantially freeof cellular material or culture medium when naturally produced, orchemical precursors or other chemicals when chemically synthesized.

Therapeutic Uses

As described herein, inventive methuosis-inducing molecules induce celldeath. In this regard, the present invention is useful in a number ofpathological applications. Certain applications are mentioned below;others will be apparent to those of ordinary skill in the art.

Therefore, according to the present invention, methuosis-inducingmolecules can be used to induce cell death of cancer cells. For example,the present invention is useful in treating cancers of the brain, lung,bladder, liver, spleen, pancreas, bone, colon, stomach, breast,prostate, ovary, central nervous system and skin. For example,glioblastoma and breast carcinoma can be treated according to thepresent invention.

Research Uses

In addition to the various pharmaceutical uses described above,methuosis-inducing compounds of the present invention have utility in avariety of research applications, e.g., in vitro assays, including, forexample, as chemical probes. Those of ordinary skill in the art willappreciate that the field of chemical genetics attempts to identifychemical agents with definable effects on biological events, pathways,or products so that these agents can be used as tools to analyze therelevant biological events, pathways, or products. Methuosis-inducingmolecules of the present invention are particularly well suited for suchstudies. Accordingly, the present invention also includes assays, e.g.,in vitro assays, utilizing the methuosis-inducing molecules of thepresent invention to analyze vacuolization, intracellular trafficking,antigen presentation, membrane fusion events, and related cellularprocesses. Furthermore, the methuosis-inducing molecules of the presentinvention can also be used in screening assays to identify secondgeneration methuosis-inducing molecules, e.g., molecules having modifiedchemical structures which function as methuosis-inducing molecules.Azide, propargyl or other forms of the methuosis-inducing molecules ofthe present invention can be used in a variety of cell-based ormolecular screening assays to identify specific protein targets thatbind to such molecules.

Formulations

As described herein, inventive methuosis-inducing compounds can beutilized in any of a variety of contexts, and can be formulatedappropriately according to known principles and technologies.

For example, the methuosis-inducing molecules can be provided insubstantially pure form, in an organic solvent such as DMSO.Alternatively or additionally, methuosis-inducing molecules can beformulated as a pharmaceutical composition, for example being combinedwith a pharmaceutically acceptable carrier. It will also be appreciatedthat certain of the compounds of present invention can exist in freeform for treatment, or where appropriate, as a pharmaceuticallyacceptable derivative thereof. According to the present invention, apharmaceutically acceptable derivative includes, but is not limited to,pharmaceutically acceptable salts, esters, salts of such esters, or aprodrug or other adduct or derivative of a compound of this inventionwhich upon administration to a patient in need is capable of providing,directly or indirectly, a compound as otherwise described herein, or ametabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith little or no undue toxicity, irritation, allergic response and thelike, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts of amines, carboxylic acids, and othertypes of compounds, are well known in the art (see, for example, Berge,et 3.1. J. Pharmaceutical Sciences, 66:1-19, 1977, incorporated hereinby reference).

The salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or separately byreacting a free base or free acid function with a suitable reagent, asdescribed generally below. For example, a free base function can bereacted with a suitable acid. Furthermore, where the compounds of theinvention carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof can, include metal salts such as alkali metal salts, e.g.sodium or potassium salts; and alkaline earth metal salts, e.g. calciumor magnesium salts. Examples of pharmaceutically acceptable, nontoxicacid addition salts are salts of an amino group formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hernisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The methuosis-inducing molecules disclosed herein can be formulated oradministered together with any other known agent having a complementarybiological effect. For example, methuosis-inducing molecules can becombined with steroids or other immunomodulating agents in order toregulate immunological events as described herein.

It will be appreciated that the compounds and compositions, according tothe method of the present invention, can be administered using anyeffective amount and any effective route of administration. Thus, theexpression “effective amount” as used herein, refers to a sufficientamount of agent to result in vacuolization and/or inhibition ofcompartment trafficking as described herein. The exact amount requiredcan vary from subject to subject, depending on the species, age, andgeneral condition of the subject, the severity of the infection, theparticular therapeutic agent, its mode of administration, and the like.The compounds of the invention are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof therapeutic agent appropriate for the patient to be treated. It willbe understood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment.

The specific therapeutically effective dose level for any particularpatient or organism can depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, gender and diet of the patient; thetime of administration, route of administration, and rate of excretionof the specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employed;and like factors well known in the medical arts.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionsof this invention can be administered to humans and other animalsorally, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by powders, ointments, or drops),bucally, as an oral or nasal spray, or the like, depending on theseverity of the infection being treated. In certain embodiments, thecompounds of the invention can be administered at dosage levels of about0.001 mg/kg to about 50 mg/kg, from about 0.01 mg/kg to about 25 mg/kg,or from about 0.1 mg/kg to about 10 mg/kg of subject body weight perday, one or more times a day, to obtain the desired therapeutic effect.It will also be appreciated that dosages smaller than 0.001 mg/kg orgreater than 50 mg/kg (for example 50-100 mg/kg) can be administered toa subject. In certain embodiments, compounds are administered orally orparenterally.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms can contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.R and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension orcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionthat, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude (poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable nonirritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They can optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype can also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art.

In such solid dosage forms the active compound can be admixed with atleast one inert diluent such as sucrose, lactose and starch. Such dosageforms can also comprise, as in normal practice, additional substancesother than inert diluents, e.g., tableting lubricants and othertableting aids such as magnesium stearate and microcrystallinecellulose. In the case of capsules, tablets and pills, the dosage formscan also comprise buffering agents. They can optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as can be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

In other embodiments of the invention, methuosis-inducing compounds, orcompositions containing them are packaged into a kit for convenientlyand effectively carrying out the methods in accordance with the presentinvention. In general, the pharmaceutical pack or kit comprises one ormore containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Such kits are especiallysuited for the delivery of solid oral forms such as tablets or capsules.Such a kit preferably includes a number of unit dosages, and can alsoinclude a card having the dosages oriented in the order of theirintended use. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarinsert, designating the days in the treatment schedule in which thedosages can be administered. Alternatively, placebo dosages, or calciumdietary supplements, either in a form similar to or distinct from thedosages of the pharmaceutical compositions, can be included to provide akit in which a dosage is taken every day. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

EXAMPLES Example 1 Methods and Materials

Test Compounds and Reagents

Compounds I, II (MIPP), and IV were purchased from ChembridgeCorporation. Compound III was purchased from TimTec, LLC. Each of thesecompounds was stored at −20° C. as a 5 mg/ml stock solution in DMSO, andthen diluted to the mentioned final concentration in cell culturemedium. Filipin and bafilomycin A1 were obtained from Sigma-Aldrich.Filipin was stored at −20° C. as a 50 mg/ml stock in DMSO andbafilomycin A1 was stored at −20° C. as a 10 μM stock in DMSO. EHT 1864was provided by Exonhit Therapeutics. z-VAD-fmk was purchased fromBachem.

Library Compounds

Compounds 1-8 used for initial screening of methuosis-inducing activity(FIG. 1) have identification numbers as follows: 1, 5224450; 2, 5224466;3, 5312531; 4, 7995005; 5, 7916760; 6, 6161388; 7, 5267766; and 8,6155359. All compounds are certified by the vendor to be at least 90%pure with NMR confirmation of structure.

General Methods

Reagents and starting materials were obtained from commercial supplierswithout further purification. Thin layer chromatography (TLC) was doneon 250 μm fluorescent silica gel 1B-F plates and visualized with UVlight. Flash column chromatography was performed using silica gel230-400 μm mesh size. Melting points (MP) are uncorrected. Nuclearmagnetic resonance (NMR) spectra were recorded on, either, a 600, 400 or200 MHz instrument. Peak locations were referenced using the residualsolvent peak (7.26 and 77.16 for CDCl3 1H and 13C, respectively, and2.50 and 39.51 for DMSO 1H and 13C). Proton coupling constants (Jvalues) and signals are expressed in hertz using the followingdesignations: s (singlet), d (doublet), br s (broad singlet), m(multiplet), t (triplet), dd (doublet of doublets) and qd (quintet ofdoublets). NMR spectra are available in the supplementary information.

Cell Culture

U251 human glioblastoma cells were purchased from the DCT TumorRepository. All other cell lines were obtained from the American TypeCulture Collection. Cell lines were passaged for fewer than six monthsprior to use. Normal human skin fibroblasts were derived from a skinbiopsy. Unless stated otherwise, cell lines were maintained inDulbecco's modified Eagle medium (DMEM) with 10% (v/v) fetal bovineserum (FBS) at 37° C. with 5% CO₂/95% air. MCF-10A cells were maintainedin DMEM+Ham's F12 (1:1) containing 5.0% horse serum, 20 ng/ml EGF, 0.5μg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 μg/ml insulin, asdescribed. Colonies of temozolomide-resistant U251 cells were selectedby maintaining the parental U251 cells in medium containing 100 μMtemozolomide for 24 days, with replenishment of the medium and drugevery three days. Colonies were then picked and plated into a 12-welldish and exposed to escalating concentrations of temozolomide. The cloneused in the present study (U251-TR) is maintained routinely in mediumwith 300 μM temozolomide. To generate cell growth curves, U251 cellswere plated in 35 mm dishes (100,000 cells/dish, FIG. 4B) and allowed toattach for 24 h. Thereafter, cells were treated with the mentionedcompounds dissolved in DMSO or with vehicle alone. At daily intervals,three parallel cultures were harvested from each group by trypsinizationand aliquots of cell suspension were counted in a Coulter Z1 particlecounter. Phase-contrast images of live cells were obtained using anOlympus IX70 inverted microscope equipped with a digital camera and SPOTimaging software.

Live Cell Imaging with Fluorescent Tracers

Lucifer yellow (LY) was purchased from Invitrogen/Molecular Probes.Labeling of endocytic compartments with this fluid-phase tracer wasperformed as previously described. Labeling of intracellular acidiccompartments with Lysotracker Red DND-99 and staining for cathepsin Bactivity with Magic Red RR were performed as described previously.ER-Tracker Blue-White DPX was used to label the endoplasmic reticulumfollowing the directions supplied by the manufacturer. Phase-contrastand fluorescent images of the living cells were acquired on an OlympusIX70 inverted microscope equipped with a digital camera and SPOT imagingsoftware or on a Nikon Eclipse TE2000U fluorescence microscope with adigital camera and NIS-Elements AR software.

Time-Lapse Microscopy

200,000 U251 cells were plated in a 35 mm glass-bottom microwell culturedish. The day after plating, the cells were treated with 10 μM MIPP inphenol red-free DMEM supplemented with 10% FBS. The dish was immediatelyplaced in a humidified Live Cell chamber equilibrated with 5% CO₂ at 37°C. The chamber was placed on the stage of an Olympus IX80 invertedmicroscope, equipped with a digital camera and Slidebook software. Thesoftware was set to automatically acquire phase-contrast images every 30sec for the designated period of time.

Treatment of Cells with Filipin, Bafilomycin A1 or EHT 1864.

To inhibit clathrin-independent endocytosis, U251 cells were washedtwice with serum-free DMEM, then pretreated for 30 min with DMEM+0.5%BSA in the presence or absence of 12 μg/ml filipin. Following thepretreatment, MIPP was added to the dishes at a final concentration of10 μM and phase-contrast images were acquired 100 min later.

To inhibit the vacuolar-type H⁺ ATPase, U251 cells were pretreated for 1h with 0.1 μM bafilomycin A1 or an equivalent volume of DMSO. At the endof the hour, 10 μM MIPP, or an equivalent volume of DMSO, was addedwithout a medium change. Phase-contrast images of the cells wereacquired 1 h after the addition of MIPP.

To determine if inhibition of the Rac1 GTPase would block theaccumulation of vacuoles, U251 cells were treated for 24 h with 10 μMMIPP in the presence or absence of 25 μM EHT 1864. The percentage ofvacuolated cells in the population was determined by scoring at least100 cells in multiple phase-contrast photomicrographs for eachcondition. Cells containing three or more phase-lucent vacuoles withdiameters ≧0.5 μm or >10 smaller vacuoles (0.1-0.5 μm) were scored aspositive. For comparison, the effect of EHT 1864 on Ras-inducedvacuolization was also determined. U251 cells were nucleofected withpCMV5-Myc-H-Ras (G12V), then immediately plated into medium with orwithout 25 μM EHT 1864. After 24 h, phase-contrast images were taken andthe number of vacuolated cells was scored as described above.

Pull-Down Assays to Measure the Activation States of GTPases

Assays for activated Rac1 and Arf6 were performed using EZ-Detect Rac1or Arf6 activation kits. These assays employ either a GST-fusion proteincontaining the p21-binding domain of p21-activated protein kinase 1(PAK1) to selectively bind active Rac1 in whole cell lysates, orGST-GGA3 (Golgi-associated gamma adaptin ear-containing Arf bindingprotein 3) to pull down active Arf6. The active Rac1 or Arf6 collectedon the glutathione beads were subjected to western blot analysis and thechemiluminescence signals were quantified using Alpha Innotech FluorChem HD2 imaging system. The values for active protein in each samplewere normalized to α-tubulin. Results were expressed as either the totalactive Rac1 or Arf6, or the ratio of the active GTPase to the total Rac1or Arf6 measured in aliquots of the cell lysate.

GST-fusion proteins were produced in E. coli BL21 (DE3) pLysS and thefusion proteins were bound to glutathione-sepharose 4B beads. Pull-downassays for active Rab5 using the GST-Rabaptin-5 beads were performed.Assays for active Rab7 were performed. For each determination celllysates were prepared from ten pooled 10 cm cultures. Monoclonalantibodies against Rab5 or Rab7 were used to probe western blots of theproteins collected on the beads. Results were quantified and normalizedas described above for Rac1 and Arf6.

Confocal Fluorescence Microscopy

U251 cells were nucleofected with pEGFP-Rab7 or pEGFP-Rab5, then platedonto coverslips in 60 mm dishes. The day after nucleofection, the cellswere treated with 10 μM MIPP. For colocalization experiments, cells thathad been treated with MIPP for 24 h were prepared for immunofluorescencemicroscopy. All primary antibodies were detected by incubation with goatanti-mouse IgG conjugated to Alexa Fluor 568. Cells were examined byconfocal microscopy using a Leica TCS SP5 system with 488- and 561-nmlaser excitation. Images were acquired with the LASAF software on thesystem.

Electron Microscopy

U251 cells were exposed to 10 μM MIPP for 48 h, then prepared forelectron microscopy. The cells were examined under a Philips CM 10transmission electron microscope.

Western Blot Analysis

The antibody for PARP was purchased from BD Biosciences. Analysismethods sued were protein determination, SDS-PAGE and western blotanalysis.

Cell Viability

Cell viability was measured using a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)-based assay. A 5 mg/ml MTT stock solution was prepared inphenol-red free RPMI 1640. Cells were seeded in 96-well plates, withfour replicate wells for each culture condition. On the day of theassay, 10 μl of the MTT solution was added directly to 10 μl in eachwell and the cells were incubated for 3-4 h at 37° C., with 5% CO₂. Atthe end of the incubation, 100 μl of MTT solvent (0.1N HCl inisopropanol, containing 0.1% NP-40) was added to each well. The plateswere incubated for an additional 5 min at 37° C., 5% CO₂, thenquantified for absorbance at 570 nm on a SpectraMax Plus 384 platereader.

For colony-forming assays, cells were plated in 100 mm dishes at 2,500(U251 and U251-TR) or 1,500 (all other cell lines) cells per dish.Beginning on the day after plating, the cells were exposed to 10 μM MIPPfor 2 days, with the medium and drug replenished after 1 day. The cellswere fed fresh medium without drug every 2 to 3 d for a period of 10-21days. To visualize the colonies, the dishes were washed with PBS, fixedfor 10 min with ice-cold 100% methanol, and stained with 1% crystalviolet in 35% methanol. After 2-3 washes with water, colonies containingat least 50 cells were counted using a dissecting microscope or aProtocol 2 colony counter.

To compare the levels of ATP in MIPP-treated glioblastoma cells versuscontrols treated with an equivalent volume of DMSO, the cells wereharvested by trypsinization and assayed using the CellTiter Glo kit fromPromega according to the manufacturer's instructions.

Statistical Significance

Statistical significance of differences in colony formation or otherparameters (e.g., GTPase activation) involving comparisons betweencontrol and MIPP-treated cells were evaluated by Student's two tailedt-test.

Example 2 Small Molecules that Induce Cytoplasmic Vacuolization

Disclosed herein is Compound I which caused a striking accumulation ofnumerous phase-lucent cytoplasmic vacuoles within 4 h when applied toU251 glioblastoma cells (FIG. 1). When added at a concentration of 1 μM,the morphological effects of Compound I were transient, with most of thevacuoles dissipating by 24 h (FIG. 1). However, at 10 μM, themorphological effects of Compound I persisted for 24 h and beyond. Asearch of the broader 700,000 compound Chembridge collection yieldedseveral additional compounds with >75% similarity to Compound I. Ofthese, 3(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (FIG. 1,Compound II), behaved similarly to Compound I when tested at 1 μM, butinduced vacuoles that were larger and more numerous than those inducedby Compound I when tested at a concentration of 10 μM (FIG. 1).

Closely related compounds with similar or identical indole ringstructures, but with variations in the second aryl ring (Compound III)or the enone linker (Compound IV), showed no vacuole-inducing activity(FIG. 1). This showed that the effects of Compounds I and II were due totheir interactions with specific intracellular targets, rather thannon-specific effects on cellular membranes or intracellular pH. CompoundIII had no vacuole-inducing activity despite the fact that it shared thecharacteristics of a Michael acceptor with Compounds I and II. Based onthese initial data, Compound II was selected for further study as asmall molecule inducer of methuosis. The same compound is compound 2 inFIG. 29 and FIGS. 36 and 48. Hereafter it will be referred to by theacronym MIPP: i.e., 3-(2-methyl-1Hindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one.

Example 3 The Origin of the Vacuoles Induced by MIPP is Consistent withMethuosis

It is further disclosed herein that the vacuoles induced by MIPP werederived from macropinosomes, since this is a hallmark feature ofmethuosis. Macropinocytosis is a form of clathrin-independentendocytosis wherein intracellular vesicles are initially generated fromprojections of the plasma membrane termed ruffles or lamellipodia, whichsurround and trap extracellular fluid. Time lapse phase-contrastmicroscopy covering the period between 13-80 min after addition of MIPPrevealed waves of macropinocytotic vesicles entering the U251 cells fromregions of active membrane ruffling. The nascent vesicles can be seencoalescing with each other to form progressively larger vacuoles withinthe cytoplasm as shown in FIG. 1. Time lapse studies performed after thefirst 95 min showed a decline in the initial burst of macropinocytoticactivity, although the vesicles already formed within the cell continuedto enlarge by undergoing occasional fusion events as shown in FIG. 2.

One of the features of methuosis is the incorporation of fluid-phasetracers like Lucifer yellow (LY) into large vacuoles that eventuallyfill the cytoplasm and disrupt the cells. Therefore, U251 cells wereincubated with LY during the first 4 h after addition of the compound toshow that the MIPP-induced vacuoles observed by phase-contrastmicroscopy were derived from macropinosomes. As shown in FIG. 3, LY wasincorporated into most of the phase-lucent vacuoles.

Macropinocytosis, which is a clathrin-independent form of endocytosis,is dependent on cholesterol-rich membrane microdomains. U251 cells werepre-incubated with or without filipin for 30 min prior to adding MIPP inorder to establish that the MIPP-induced vacuoles were derived fromclathrin-independent compartments. As shown in FIG. 4, cells treatedwith MIPP in the absence of filipin formed numerous vacuoles within thefirst 100 min after addition of the compound. In contrast, the cellstreated with filipin failed to show the typical morphological responseto MIPP. Thus, the majority of the vacuoles induced by MIPP are shown tooriginate from clathrin-independent macropinosomes, consistent with themechanism of methuosis.

Example 4 Relationship of MIPP-Induced Vacuoles to Other SubcellularCompartments

In Ras-induced methuosis, the accumulated vacuoles eventually acquiresome characteristics of late endosomes, but remain separate from theendoplasmic reticulum and the lysosomal or autophagosomal degradativecompartments. The data disclosed herein show that the vacuoles inducedby MIPP fit this profile. Live cell imaging of the phase-lucent vacuolesinduced by MIPP showed no overlap with compartments labeled withLysoTracker Red, which identifies lysosomes based on their acidic pH, orMagic Red (RR), a cell permeable substrate that fluoresces when cleavedby the lysosomal protease, cathepsin B, as shown in FIG. 3. Nor did thevacuoles incorporate ER-Tracker, a marker for the endoplasmic reticulum,as shown in FIG. 3.

Bafilomycin A1 (Baf-A) is a specific inhibitor of the vacuolar-typeH⁺-ATPase, which plays an important role in the maintenance of endosomalmembrane potential and luminal pH. Inhibition of H⁺-ATPase with Baf-Aimpedes the formation of vesicular intermediates between early and lateendosomes. Moreover, Baf-A strongly inhibits homotypic endosome fusionin vitro.

As shown in FIG. 5, short-term incubation of U251 cells with Baf-A byitself had no morphological effect on the cells, but the inhibitorcompletely abrogated the ability of the cells to generate vacuoles whenthey were exposed to MIPP. The effect of Baf-A is shown to bespecifically related to its inhibition of the proton pump and themaintenance of endosomal membrane potential rather than generalalkalinization of the endosomal compartment, since incubating cells with1-5 mM ammonium chloride did not replicate the effects of Baf-A (datanot shown).

Further disclosed herein is an analysis of distribution of markers forearly and late endosomes and autophagosomes in cells treated with MIPP.Confocal microscopy demonstrated that by 24 h almost all of the vacuolescontained late endosomal markers, GFP-Rab7 and LAMP1, but showed littleor no overlap with early endosomes (EEA1), recycling endosomes (Rab11),or autophagosomes (LC3II), as shown in FIG. 6. The presence of the lateendosome marker, Rab7, can be detected in membranes of the vacuoles assoon as 30 min after the addition of MIPP, whereas the early endosomemarker, Rab5, is generally absent from the majority of the vacuoles, asshown in FIG. 22. These data show that cellular vacuolization induced byMIPP involves fusion of nascent macropinosomes to form large vacuolesthat can rapidly mature to acquire some characteristics of lateendosomes, but cannot merge with lysosomes or autophagosomes.

Example 5 MIPP Affects the Activities of Endosomal Rab GTPases, but notRac1 or Arf6

To begin to explore the molecular mechanism(s) through which MIPP causesendosomal vacuolization, the inventors considered possible parallelswith the mechanism of methuosis triggered by over-expression ofactivated H-Ras. In the latter case, it is shown that development of thevacuolar phenotype requires activation of the Rac1 GTPase, with aconcomitant reduction in the activation state of another GTPase, Arf6.However, when fusion proteins that bind specifically to the activatedforms of Rac1 or Arf6 were used in pull-down assays to measure theactivation states of these GTPases, it was shown that treatment of cellswith MIPP had no significant effects on the amounts of active Rac1 (FIG.7) or Arf6 (FIG. 8) at either 4 h or 24 h after addition of thecompound. When U251 cells were incubated with MIPP in the presence ofEHT 1864, a highly specific Rac inhibitor, there was no detectableeffect on vacuole formation (FIG. 23). These data show that themechanism of vacuolization induced by MIPP is different from thatinduced by Ras, in that it does not depend on activation of the Rac1signaling pathway.

In light of the striking effects of MIPP on the clathrin-independentendosomal compartment, the inventors next focused on the Rab5 and Rab7GTPases, which are known to function in early and late endosomaltrafficking steps, respectively. The active GTP-bound forms of theseproteins were measured in pull-down assays using GST-fusion constructscontaining the Rab binding domains of rabaptin-5, for Rab5, or RILP, forRab7. As shown in FIG. 9, MIPP caused a striking decline in the amountof active Rab5 at both 4 h and 24 h after treatment. In contrast, MIPPhad the opposite effect on Rab7, with the amount of active Rab7 morethan doubling by 24 h, as shown in FIG. 10.

The changes in active Rab5 and Rab7 at 24 h were moderated somewhat (butstill significant) when the results were normalized to the total Rab5 orRab7 pools. This is due to the fact that the total amounts of Rab5 andRab7 in the MIPP treated cells increased substantially by 24 h. It isimportant to note that the changes in the Rab activity were observedonly in cells treated with the vacuole-inducing compound, MIPP. Forexample, in cells treated with the related but inactive Compound III(see FIG. 1), there was no change in the activation state of Rab7 (FIG.11). This data shows that the vacuolization of macropinosome-derivedendocytic compartments in MIPP-treated cells is related to oppositechanges in the pools of active Rab5 and Rab7.

Example 6 MIPP Induces Non-Apoptotic Cell Death with the Characteristicsof Methuosis

Further disclosed herein are a series of studies conducted to determinehow closely the sequelae of MIPP treatment match the cell deathphenotype associated with methuosis. The initial accumulation ofvacuoles in glioblastoma cells undergoing Ras-induced methuosis isfollowed by a decline in cellular ATP levels, cell rounding, anddetachment of cells from the substratum. Cell death ensues as thevacuoles expand to fill most of the cytoplasmic space and cell membraneintegrity is disrupted. Characteristically, these alterations are notaccompanied by morphological changes typical of apoptosis, such asnuclear chromatin condensation, nuclear fragmentation or cell shrinkage.Although caspase activation can be detected by examining PARP cleavage,cell death by methuosis cannot be prevented by treatment with caspaseinhibitors. The data shown in FIGS. 12-17 show that the form of celldeath induced by MIPP in U251 glioblastoma cells shares all of thesecharacteristics.

As shown in FIG. 12, both of the vacuole-inducing compounds, MIPP andCompound I, caused a marked decrease in the relative cell viability,measured by MTT assay, during the first two days of treatment. Incontrast, the structurally related compounds III and IV that did notcause cellular vacuolization (FIG. 1) had little effect on cellgrowth/viability during the same period (FIG. 12). By the second dayafter addition of MIPP, the cells exhibited a significant decrease inATP, indicative of metabolic compromise (FIG. 13). Further, there was asubstantial increase in the number of rounded and detached cells in theMIPP-treated cultures between days 2 and 3 as shown in FIG. 14. Cellsthat had been treated with 10 μM MIPP for 2 days were non-viable incolony-forming assays, as shown in FIG. 15.

Further evaluation of the MIPP-treated cells by electron microscopyrevealed that both the attached and detached cells contained numerouslarge, mostly empty, vacuoles bounded by a single membrane (FIG. 16).These structures were indistinguishable morphologically from thevacuoles induced by activated Ras. In the detached cells, the vacuoleshad expanded to the point where they displaced much of the cytoplasmicvolume and, in many cases, the plasma membrane was disrupted (FIG. 16,arrows). Even in highly vacuolated cells that were on the verge oflysis, the nuclear chromatin remained diffuse and the nuclear membranewas intact (FIG. 16). This was showing of a non-apoptotic deathmechanism. The dying cells treated with MIPP showed caspase activation(i.e., cleavage of full-length PARP to an 82 kDa fragment). However,even though zVAD-fmk was able to block this cleavage, it did not preventthe loss of viability in the MIPP-treated cells. When combined with thelack of a morphological signature for apoptosis, this data shows thatMIPP-induced cell death is independent of caspase activation and is dueto physical disruption of the highly vacuolated cells.

Example 7 MIPP Produces Similar Effects in a Broad Spectrum of CancerCells, Including Drug-Resistant Glioblastoma

Also disclosed herein is an analysis of the effects of the compound inseveral other cell lines. These include an additional glioma cell line(LN229), osteosarcoma cells (U2OS), and breast (MCF7), colon (SW480) andpancreatic (PANC-1) carcinoma cells. Similar to the results with U251cells, 10 μM MIPP induced dramatic cytoplasmic vacuolization in all ofthe cell lines, as shown in FIG. 26. Although there were somedifferences in sensitivity, relative cell viability determined by MTTassays was generally reduced by 50-90% in all of the cancer cell linestreated with MIPP, as shown in FIG. 27. Colony forming assays confirmedthat exposure to MIPP for 2 days significantly reduced long-term cellsurvival in all cases, as shown in FIG. 28.

Further disclosed herein is an examination of the effects of MIPP onnormal human skin fibroblasts and an established mammary epithelial cellline (MCF-10A). Although these cell lines also underwent extensivecytoplasmic vacuolization, the reductions in cell viability (30% forMCF-10A and 40% for fibroblasts) were more moderate than what theinventors observed for the cancer cell lines, as shown in FIG. 27.

Further disclosed herein are derived temozolomide-resistant clones fromthe U251 glioblastoma cell line. The survival study depicted in FIG. 18shows an example of one such clone (U251-TR), which was highly resistantto temozolomide in comparison to the parental U251 cell line. WhenU251-TR cells were treated with 10 μM MIPP, they underwent extensivevacuolization identical to that observed in the parental U251 cells, asshown in FIG. 19. MTT dose-response curves showed that both the parentaland temozolomide-resistant cells were sensitive to MIPP over a 2-dayperiod (FIG. 20), although the IC₅₀ value for the resistant cells (6.0μM) was higher than for the parental cells (3.5 μM). As in the case ofthe parental U251 cells (FIG. 15), treatment of the U251-TR cellsresulted in a significant decline in survival, assessed by colonyforming assays (FIG. 21). Similar results were obtained with additionaltemozolomide-resistant clones (not shown).

Example 8 Comparisons of MIPP and MOMIPP

The foregoing SAR studies identified compound 19 as the most potent inthe series for inducing methuosis. Hereinafter this new lead compound isreferred to as MOMIPP; 3-(5-methoxy,2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one. Enhancedactivity of MOMIPP versus MIPP was confirmed in studies with U251glioblastoma cells, using MTT viability assays, cell growth assays,morphological assessment, and colony forming assays to compare the twocompounds. FIG. 37D shows the dose-response curves for the effects ofthe two drugs on cell viability.

Each compound was added at the designated concentration for two days,with medium and compound replenished after the first day. The IC₅₀ forMOMIPP was 1.9 μM, versus 4.8 μM for MIPP. To obtain a measure of therelative stability and duration of the effects of each compound, theireffects on cell growth and survival was tested by counting the number ofcells in parallel cultures treated for three days with 2.5 μM, 5 μM, or10 μM compound, as shown in FIG. 38. Unlike the viability studies inFIGS. 36, 37D and 48, in this instance each compound was added at thebeginning of the experiment and the medium was not replenished for theduration. Under these conditions, MOMIPP was clearly more effective thanMIPP in reducing cell growth. The reduction of cell number in thecultures treated with MOMIPP coincided with massive early vacuolizationof the cells and loss of nonviable cells from the substratum (FIG. 39).In contrast, the cells treated with MIPP initially underwentvacuolization on days 1 and 2, but tended to recover, especially at thelower concentrations of the compound (FIG. 39).

The data from these studies demonstrate that MOMIPP has a much moresustained effect than MIPP on cell morphology and cell viability.Further, dose-response comparisons of MIPP versus MOMIPP using colonyforming assays was performed to assess effects on long-term cellsurvival. As shown in FIG. 40, both MIPP and MOMIPP reduced colonyformation of U251 glioblastoma cells when the cells were treated for 2days, although MOMIPP was much more potent than MIPP. Both compoundsalso reduced colony formation when cells were treated for only 4 hours,although higher concentrations of compound were required. Again, MOMIPPwas more effective than MIPP (FIG. 40).

Example 9 MOMIPP Kills Drug Resistant Glioblastoma Cells

Further disclosed herein is data showing that MIPP can effectivelyinduce methuosis and kill temozolomide-resistant U251 glioblastoma cells(FIGS. 19-21). MOMIPP can induce methuosis and reduce the viability oftemozolomide-resistant glioblastoma cells (FIGS. 41A-41B) anddoxorubicin-resistant breast cancer cells (FIGS. 41C, 41D, 45 and 46).MOMIPP and structurally-related compounds are shown to be usefultherapeutic agents for inducing non-apoptotic cell death (i.e.,methuosis) in drug-resistant glioblastoma cells.

Example 10 MOMIPP Kills a Broad Spectrum of Glioblastoma Cells,Including Glioblastoma Stem Cells

The ability of MOMIPP to induce methuosis in GBM cells is not restrictedto the U251 cell line. The compound induced characteristic cytoplasmicvacuolization in several additional GBM cell lines: U87MG, LN229, SF295and T98G, as shown in FIG. 42. In all cases, 10 μM MOMIPP causedsignificant (p<0.005) loss of cell viability detected by MTT assay after2 d, with no replenishment of the drug (FIG. 42). Long term survival,measured by colony forming assays, was severely attenuated in GBM cellstreated with 10 μM MOMIPP for 2 d. (FIG. 43). The affected cell lineshave different genetic profiles. For example, U87MG is wt for p53, whilethe others have mutations in p53. LN229 is wt for PTEN, while the othersare null or mutant at the PTEN locus. None of the cell lines harbor Rasmutations. Thus, it is shown that the ability of MOMIPP to inducemethuosis in these GBM cell lines is consistent the compound acting atthe level of the endosomal trafficking machinery, independent ofvariations in tumor-suppressor or oncogenic signaling pathways. GMB6 waschosen because it retains amplification of EGFRvIII and exhibitsinvasive behavior in vivo. The acute response of GBM6 to MOMIPP was thesame as the established cell lines, as shown in FIG. 42, and colonyformation was strongly inhibited (FIG. 43). Studies with CD133⁺ stemcells isolated from xenografts of primary human GBM were carried out.When treated with MOMIPP, these cells exhibited the typical vacuolarmorphology of methuosis, with reduced viability after 2 d (FIG. 42).When CD133⁺ stem cells isolated directly from a primary GBM specimenwere treated with MOMIPP, the compound significantly inhibited colonyformation at concentrations as low as 2.5 μM (FIG. 44).

Example 11 MOMIPP Kills Drug Resistant Breast Cancer Cells

MOMIPP can induce methuosis and reduce the viability ofdoxorubicin-resistant breast cancer cells, as is shown by severecytoplasmic vacuolization in FIG. 45, reduced metabolic viability inFIG. 46 and decreased ability to form colonies in FIG. 47. Sensitivityof the drug resistant breast cancer cells to MOMIPP was comparable tothe parental MCF7 cell line. MOMIPP and structurally-related compoundsare shown to be useful therapeutic agents for inducing non-apoptoticcell death (i.e., methuosis) in drug-resistant breast cancer cells.

Example 12 MOMIPP Synthesis

5-Methoxy-2-methyl-1H-Indole-3-carboxaldehyde

To an oven dried 25 mL round bottom flask kept under a nitrogenatmosphere and maintained at 0° C., 0.069 mL (0.744 mmol, 1.2 equiv.) ofphosphorous oxychloride (POCl₃) was added to 0.25 mL dimethylformamide(DMF). After stirring for ten minutes, 100 mg of5-methoxy-2-methyl-indole (0.620 mmol, 1.0 equiv.) dissolved in 0.75 mLDMF was added dropwise. After 45 minutes of stirring at 0° C., 1N NaOHwas added (5 mL), forming a white precipitate which was extracted withethyl acetate (3×10 mL). The extracts were washed with brine, dried withNa₂SO₄, filtered, concentrated, and dried under vacuum, affording 105 mgof white solid (90%). ¹H NMR (600 MHz, d₆-DMSO): δ 11.875 (s, 1H),10.009 (s, 1H), 7.570-7.566 (d, J=2.4, 1H), 7.282-7.268 (d, J=8.4, 1H),6.797-6.778 (dd, J₁=9.0, J_(z)=2.4, 1H), 3.764 (s, 3H), 2.646 (s, 3H).¹³C NMR (150 MHz, d₆-DMSO): δ 184.1, 155.5, 148.6, 130.0, 126.4, 113.7,112.1, 111.8, 102.3, 55.3, 11.6. Melting point: 187-193° C. TLC (in 4:1ethyl acetate:hexanes) R_(f)=0.37. Elemental analysis calculated forC₁₁H₁₁NO₂: C, 69.83; H, 5.86; N, 7.40. found: C, 69.95; H, 5.95; N,7.25.

MOMIPP,trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one

To an oven dried 25 mL round bottom flask kept under a nitrogenatmosphere, 80 mg of 5-methoxy-2-methyl-1H-Indole-3-carboxaldehyde (0.42mmol, 1.0 equiv.) was added, followed by 2 mL anhydrous methanol, 59 μLof 4-acetylpyridine (0.53 mmol, 1.25 equiv.) and 47 μL of piperidine(0.46 mmol, 1.1 equiv.) and refluxed for 16 hours. Upon cooling thereaction to room temperature, an orange precipitate formed, which wasfiltered under vacuum and washed with cold methanol. The orange solidwas dried under vacuum, yielding 110 mg (90%) of an orange solid. ¹H NMR(600 MHz, d₆-DMSO): δ 11.909 (s, 1H), 8.812-8.802 (dd, J=4.2, J₂=1.8,2H), 8.097-8.072 (d, J=15, 1H), 7.947-7.937 (dd, J=4.2, J₂=1.8, 2H),7.434-7.430 (d, J=2.4, 1H), 7.374-7.349 (d, J=15, 1H), 7.315-7.304 (d,J=8.4, 1H), 6.851-6.833 (dd, J₁=8.4, J2=2.4, 1H), 3.860 (s, 3H), 2.572(s, 3H). ¹³C NMR (150 MHz, d₆-DMSO): δ 188.0, 155.2, 150.6, 145.8,145.1, 136.6, 131.0, 126.6, 121.4, 112.8, 112.3, 110.9, 109.3, 103.5,55.6, 12.2. Melting point: 255-257° C. TLC (in 4:1 ethylacetate:hexanes) R_(F)=0.16. Elemental analysis calculated forC₁₈H₁₆N₂O₂: C, 73.95; H, 5.52; N, 9.58. found: C, 73.76; H, 5.46; N,9.47.

Example 13 Structure Activity Relationship Compound Synthesis2-Methylindole-3-carboxaldehyde (compound 2a)

To a dried 300 mL two-neck round bottom flask under argon at 0° C.,N,N-dimethylformamide (3 mL) was added, followed by POCl3 (1.05 mL, 11.3mmol). After stirring for ten minutes, 2-methylindole (1.23 g, 9.37mmol) dissolved in DMF (6 mL) was added dropwise via an addition funnelunder argon. After two hours, 1N NaOH (70 mL) was added slowly, uponwhich a white precipitate formed. The solid was filtered and dried undervacuum, yielding 1.32 g (89%) of white solid. ¹H NMR (600 MHz, d6-DMSO):δ 11.998 (s, 1H, N—H), 10.050 (s, 1H, CHO), 8.044-8.032 (d, J=7.2, 1H,indole-4H), 7.388-7.375 (d, J=7.8, 1H, indole-7H), 7.183-7.135 (m, 2H,indole-5,6H), 2.679 (s, 3H, methyl). 13CNMR (150 MHz, d6-DMSO): δ 184.3,148.6, 135.4, 125.6, 122.7, 121.9, 120.0, 113.7, 111.4, 11.5. MeltingPoint: 195-200° C. TLC (ethyl acetate:hexanes 4:1) Rf=0.39. Elementalanalysis calculated for C10H9NO: C, 75.45; H, 5.70; N, 8.80. found: C,75.23; H, 5.70; N, 8.93.

trans-3-(2-Methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 2)

To a dried 500 mL round bottom flask under argon,2-methylindole-3-carboxaldehyde (400 mg, 2.51 mmol) was dissolved inanhydrous MeOH (10 mL). 4-Acetyl-pyridine (305 μL, 2.76 mmol, 1.1equiv.) and piperidine (82 μL, 0.83 mmol) were added and the reactionwas stirred under reflux. A red-orange precipitate gradually formed, andafter twelve hours this solid was isolated by filtration, rinsed withchilled methanol and dried under vacuum, producing 458 mg (69%) ofyellow solid. NMR: 1H NMR (600 MHz, d6-DMSO): δ 12.017 (s, 1H, N—H),8.816-8.806 (dd, J1=4.5, J2=2.1, 2H, pyr-2,6H), 8.121-8.095 (d, J=15.6,1H, C═CH), 8.077-8.063 (m, 1H, indole-4H), 7.975-7.965 (dd, J1=4.5,J2=1.5, 2H, pyr-3,5H), 7.489-7.464 (d, J=15.6, 1H, C═CH), 7.415-7.400(m, 1H, indole-7H), 7.220-7.200 (m, 2H, indole-5,6H), 2.596 (s, 3H,methyl). 13C NMR (150 MHz, d6-DMSO): δ 188.0, 150.7, 145.6, 144.9,139.5, 136.3, 125.8, 122.4, 121.50, 121.46, 120.4, 113.1, 111.7, 109.4,11.9. Melting Point: 268-272° C. TLC: (ethyl acetate:hexanes 4:1)Rf=0.18. Elemental analysis calculated for C17H14N20: C, 77.84; H, 5.38;N, 10.68. found: C, 77.96; H, 5.28; N, 10.59.

trans-3-(1H-Indol-3-yl)-1-phenyl-2-propen-1-one (compound 9)

In a dried, 25 mL round bottom flask under argon,indole-3-carboxaldehyde (300 mg, 2.07 mmol) was dissolved in anhydrousmethanol (8 mL). Acetophenone (240 μL, 2.07 mmol) and piperidine (100μL, 1.00 mmol) was added. The reaction was stirred under reflux for 18hours. 10% Acetic acid was added (10 mL), precipitating 248 mg of acrude yellow solid. This was recrystallized in 100% EtOH, filtered, anddried under vacuum, yielding a pure, yellow solid (198 mg, 39%, 247.29MW). 1H NMR (600 MHz, d6-DMSO): δ 8.133-8.114 (m, 3H, phenyl-2,6H,indole-2H), 8.095-8.081 (m, 1H, indole-4H), 8.075-8.049 (d, J=15.6, 1H,C═CH), 7.668-7.642 (d, J=15.6, 1H, C═CH), 7.655-7.629 (m, 1H,phenyl-4H), 7.581-7.556 (t, J=1.5, 2H, phenyl-3,5H), 7.504-7.490 (m, 1H,indole-7H), 7.254-7.231 (m, 2H, indole-5,6H). ¹³C NMR (150 MHz,d6-DMSO): δ 188.8, 139.1, 138.5, 137.5, 133.4, 132.4, 128.7, 128.2,125.1, 122.8, 121.2, 120.4, 115.3, 112.8, 112.5. Melting point: 167-170°C. TLC (in 2:1 ethyl acetate:hexanes) Rf=0.48. Elemental analysiscalculated for C17H13NO: C, 82.57; H, 5.30; N, 5.66. found: C, 82.19; H,5.35; N, 5.63.

trans-3-(1H-Indol-3-yl)-1-(2-pyridinyl)-2-propen-1-one (compound 10)

Indole-3-carboxaldehyde (200 mg, 1.38 mmol) was added to a dried 100 mLround bottom flask under argon and anhydrous methanol (8 mL) was added.2-Acetyl-pyridine (232 μL, 2.07 mmol) and piperidine (69 μL, 0.7 mmol)were added and the reaction was stirred under reflux for 24 hours, afterwhich still no precipitate had formed (AcOH did not lead toprecipitation). The crude reaction mixture was concentrated and directlyapplied to a silica column for chromatography (ethyl acetate:hexanes1:1). The product was partially separated from the aldehyde startingmaterial (some product coeluted with aldehyde and was discarded), and 80mg (23%) of purified product was isolated. NMR: 1H NMR (600 MHz,d6-DMSO): δ 11.989 (s, 1H, N—H), 8.831-8.819 (m, pyr-H2), 8.220-8.194(d, J=15.6, 1H, C═CH), 8.154-8.105 (m, 3H, C═CH, indole-H2, pyr-H3),8.052-8.027 (m, 1H, pyr-H4), 7.976-7.962 (m, 1H, indole-H4), 7.679-7.656(m, 1H, pyr-H5), 7.522-7.508 (m, 1H, indole-H7), 7.285-7.262 (m, 2H,indole-H5,6). 13C NMR (150 MHz, d6-DMSO): δ 188.2, 154.3, 149.1, 139.3,137.7, 137.6, 134.2, 127.1, 125.1, 122.3, 122.2, 121.4, 120.1, 114.3,113.1, 112.7. Melting Point: 141-145° C. TLC: (ethyl acetate:hexanes4:1) Rf=0.40. Elemental analysis calculated for C16H12N2O.0.1C4H8O2: C,76.62; H, 5.02; N, 10.90. found: C, 76.41; H, 5.03; N, 10.80.

trans-3-(1H-Indol-3-yl)-1-(3-pyridinyl)-2-propen-1-one (compound 11)

Indole-3-carboxaldehyde (100 mg, 0.69 mmol) was added to a dried 100 mLround bottom flask and dissolved in anhydrous methanol (5 mL).3-Acetyl-pyridine (113 μL, 1.03 mmol, 1.5 equiv.) and piperidine (69 μL,0.7 mmol) were added and the reaction stirred under reflux. After twelvehours, the reaction was cooled to room temperature, upon which aprecipitate formed. The solid was filtered, yielding 54 mg. However,there was significant aldehyde present on a crude NMR. This mixture wasdry-loaded onto silica and purified by column chromatography (methylenechloride:methanol 9:1), producing 33 mg pure, yellow solid (19%). NMR:1H NMR (600 MHz, d6-DMSO): δ 12.004 (s, 1H, N—H), 9.292-9.289 (d, J=1.8,1H, pyr-H2), 8.808-8.797 (dd, J1=4.8, J2=1.8, 1H, pyr-H6), 8.467-8.447(dt, J1=7.8, J2=2.1, 1H, pyr-H4), 8.174 (s, 1H, indole-H2), 8.154-8.140(dd, J1=6.6, J2=1.8, 1H, indole-H4), 8.120-8.095 (d, J=15.0, 1H, C═CH),7.665-7.639 (d, J=15.6, 1H, C═CH), 7.607-7.585 (m, 1H, pyr-H5),7.506-7.492 (dd, J1=6.9, J2=1.5, 1H, indole-H7), 7.268-7.223 (qd,J1=6.6, J2=1.5, 1H, indole-H5,6). 13C NMR (150 MHz, d6-DMSO): δ 187.9,152.7, 149.3, 139.9, 137.6, 135.7, 134.1, 133.7, 125.1, 123.9, 122.9,121.3, 120.7, 115.0, 112.9, 112.5. Melting Point: 192-194° C. TLC(methylene chloride:methanol 9:1) Rf=0.26. Elemental analysis calculatedfor C16H12N2O: C, 77.40; H, 4.87; N, 11.28. found: C, 77.00; H, 4.80; N,11.12.

trans-3-(1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (compound 12)

In a dried, 25 mL round bottom flask under argon,indole-3-carboxaldehyde (300 mg, 2.07 mmol) was dissolved in anhydrousmethanol (8 mL). 4-Acetyl-pyridine (229 μL, 2.07 mmol) and piperidine(100 μL, 1.00 mmol) were added. The reaction was stirred under reflux;gradually, yellow product recipitated during the reaction. After 14hours, the reaction was cooled to room temperature, filtered and washedwith chilled methanol and hexanes. Drying under vacuum for two hoursyielded a pure, yellow solid (343 mg, 67%). 1H NMR (600 MHz, d6-DMSO): δ8.826-8.816 (dd, J1=4.2, J2=1.8, 2H, pyr-H2,6), 8.186-8.182 (d, J=2.4,1H, indole-H2), 8.128-8.117 (m, 1H, indole-H4), 8.121-8.095 (d, J=15.6,1H, C═CH), 7.978-7.968 (dd, J1=4.2, J2=1.8, 2H, pyr-H3,5), 7.592-7.566(d, J=15.6, 1H, C═CH), 7.512-7.498 (m, 1H, indole-H7), 7.267-7.243 (m,2H, indole H-5,6). 13C NMR (150 MHz, d6-DMSO): δ 188.4, 150.7, 144.7,140.9, 137.6, 134.6, 125.0, 123.0, 121.5, 121.4, 120.6, 114.6, 112.9,112.6. Melting point: 266-268′C. TLC (in 4:1 ethyl acetate:hexanes)Rf=0.23. Elemental analysis calculated for C16H12N2O: C, 77.40; H, 4.87;N, 11.28. found: C, 77.35; H, 4.84; N, 11.23.

trans-3-(5-Methoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 13)

In a dried, 25 mL round bottom flask under argon,5-methoxyindole-3-carboxaldehyde (100 mg, 0.57 mmol) was dissolved inanhydrous methanol (3 mL). 4-Acetyl-pyridine (63 μL, 0.57 mmol) andpiperidine (30 μL, 0.3 mmol) were added. The reaction was stirred underreflux, during which a crude yellow solid precipitated. After 15 hours,10% acetic acid (10 mL) was added to further the precipitation. Thesolid was filtered and dried under vacuum, yielding a pure, yellow solid(121 mg, 76%). 1H NMR (600 MHz, d6-DMSO): δ 8.821-8.811 (dd, J1=4.5,J2=1.5, 2H, pyr-H2,6), 8.163-8.159 (d, J=2.4, 1H, indole-H2),8.117-8.091 (d, J=15.6, 1H, C═CH), 7.953-7.943 (dd, J1=4.5, J2=1.5, 2H,pyr-H3,5), 7.532-7.506 (d, J=15.6, 1H, C═CH), 7.489-7.485 (d, J=2.4, 1H,indole-H4), 7.405-7.391 (d, J=8.4, 1H, indole-H7), 6.902-6.883 (dd,J1=9.0, J2=2.4, 1H, indole-H6), 3.866 (s, 3H, methyl). 13C NMR (150 MHz,d6-DMSO): δ 188.4, 155.2, 150.7, 144.9, 140.9, 134.2, 132.4, 126.0,121.5, 114.3, 113.3, 112.7, 112.4, 102.4, 55.6. Melting point: 235-237°C. TLC (in 4:1 ethyl acetate:hexanes) Rf=0.20. Elemental analysiscalculated for C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. found: C, 73.55;H, 5.00; N, 10.04.

trans-3-(5-Phenylmethoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 14)

In a dried, 25 mL round bottom flask under argon,5-benzyloxyindole-3-carboxaldehyde (100 mg, 0.40 mmol) was dissolved inanhydrous methanol (3 mL). 4-Acetyl-pyridine (75 μL, 0.68 mmol) andpiperidine (20 μL, 0.2 mmol) were added. The reaction was stirred underreflux, during which a crude yellow solid precipitated. The solid wasfiltered, rinsed with cold methanol and dried under vacuum. This crudeproduct (107 mg) was purified from residual aldehyde by columnchromatography in ethyl acetate:hexanes (1:1-3:1 gradient), yieldingpure, yellow solid (70 mg, 49%). 1H NMR (600 MHz, d6-DMSO): δ8.837-8.827 (dd, J1=4.5, J2=1.5, 2H, pyr-2,6H), 8.148-8.143 (d, J=3.0,1H, indole-2H), 8.095-8.069 (d, J=15.6, 1H, C═CH), 7.932-7.922 (dd,J1=4.5, J2=1.5, 2H, pyr-3,5H), 7.556-7.553 (d, J=1.8, 1H, indole-4H),7.523-7.511 (d, J=7.2, 2H, phenyl-2,6H), 7.460-7.434 (d, J=15.6, 1H,C═CH), 7.411-7.370 (m, 3H, phenyl-3,5H, indole-7H), 7.330-7.306 (t,J=7.2, 1H, phenyl-4H), 6.979-6.961 (dd, J1=8.4, J2=2.4, 1H, indole-6H),5.252 (s, 2H, methylene). 13C NMR (150 MHz, d6-DMSO): 188.4, 154.1,150.7, 144.9, 140.9, 137.7, 134.6, 132.5, 128.4, 127.7, 127.6, 125.8,121.5, 114.2, 113.3, 113.1, 112.7, 104.1, 69.8. Melting point: 218-221°C. TLC (in 4:1 ethyl acetate:hexanes) Rf=0.26. Elemental analysiscalculated (for C23H19N2O2. 0.2 C6H14. 0.05 H2O): C, 78.02; H, 5.93; N,7.52. found: C, 77.69; H, 5.62; N, 7.24.

trans-3-(5-Hydroxy-1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 15)

To a dried two-neck 250 mL round bottom flask under argon at −40° C.,compound 13 (352 mg, 0.99 mmol) was partially dissolved in CH2Cl2 (30mL). BBr3 (10 mL, 1.0 M in CH2Cl2, 10 mmol) was added dropwise via anaddition funnel under argon. After four hours the reaction was pouredonto ice and treated with 5 N NaOH until pH≈12. The aqueous solution wasisolated and treated with 5 N HCl until pH 7, forming a brownprecipitate which was extracted with ethyl acetate (3×40 mL). Extractswere combined, dried with Na2SO4, filtered, concentrated and dried undervacuum to yield 158 mg of an orange solid (61%). NMR: 1H NMR (600 MHz,d6-DMSO): 11.840 (s, 1H, N—H), 9.109 (s, 1H, O—H), 8.826-8.816 (dd,J1=4.5, J2=1.5, 2H, pyr-2,6H), 8.063-8.058 (d, J=3.0, 1H, indole-2H),8.048-8.022 (d, J=15.6, 1H, C═CH), 7.902-7.892 (dd, J1=4.5, J2=1.5, 2H,pyr-3,5H), 7.349-7.346 (d, J=1.8, 1H, indole-4H), 7.307-7.293 (d, J=8.4,1H, indole-H7), 6.762-6.744 (dd, J1=8.4, J2=2.4, 1H, indole-H6). 13C NMR(150 MHz, d6-DMSO): 188.2, 152.9, 150.6, 145.0, 141.4, 134.8, 131.7,126.0, 121.3, 113.6, 113.1, 112.7, 112.3, 104.9. Melting Point: 262-268°C. TLC (in ethyl acetate:hexane 4:1) Rf=0.35. Elemental analysiscalculated for C16H12N2O2.0.1H2O: C, 72.22; H, 4.62; N, 10.53. found: C,72.21; H, 4.42; N, 10.26.

trans-3-(5-Methoxy-1H-indol-3-yl)-1-(3-pyridinyl)-2-propen-1-one(compound 16)

In a dried, 50 mL round bottom flask under argon,5-methoxyindole-3-carboxaldehyde (100 mg, 0.57 mmol) was dissolved inanhydrous methanol (4 mL). 3-Acetyl-pyridine 63 μL, 0.57 mmol) andpiperidine (30 μL, 0.30 mmol) were added. The reaction was stirred underreflux, during which a crude orange solid precipitated. After twentyhours, the solid was isolated by vacuum filtration, rinsed with chilledmethanol and dried under vacuum, yielding a pure, orange solid (98 mg,60%). 1H NMR (600 MHz, d6-DMSO): δ 11.891 (s, 1H, N—H), 9.276-9.273 (d,J=1.8, 1H, pyr-2H), 8.798-8.790 (m, 1H, pyr-6H), 8.440-8.421 (m, 1H,pyr-4H), 8.155-8.150 (d, J=3.0, 1H, indole-2H), 8.113-8.088 (d, J=15.0,1H, C═CH), 7.607-7.582 (d, J=15.0, 1H, C═CH), 7.598-7.584 (m, 1H,pyr-5H), 7.500-7.497 (d, J=1.8, 1H, indole-4H), 7.397-7.383 (d, J=8.4,1H, indole-7H), 6.892-6.874 (dd, J1=8.4, J2=2.4, 1H, indole-6H), 3.867(s, 3H, methyl). 13C NMR (150 MHz, d6-DMSO): δ 187.9, 155.1, 152.6,149.3, 139.9, 135.7, 133.8, 133.7, 132.3, 126.1, 123.9, 114.7, 113.2,112.7, 112.4, 102.6, 55.6. Melting point: 169-173° C. TLC (in 4:1 ethylacetate:hexanes) Rf=0.17. Elemental analysis calculated C17H14N2O2: C,73.37; H, 5.07; N, 10.07. found: C, 73.09; H, 5.10; N, 10.01.

trans-3-(5-Methoxy-1H-indol-3-yl)-1-(pyrazine)-2-propen-1-one (compound17)

In a dried, 25 mL round bottom flask under argon,5-methoxyindole-3-carboxaldehyde (75 mg, 0.43 mmol) was dissolved inanhydrous methanol (4 mL). Acetyl-pyrazine (52 mg, 0.43 mmol) andpiperidine (23 μL, 0.23 mmol) were added. The reaction was stirred underreflux, during which a crude, yellow solid precipitated. After threehours, the solid was isolated by vacuum filtration, rinsed with chilledmethanol and dried under vacuum. This crude product was recrystallizedin EtOH (8 mL) to remove residual aldehyde, yielding a pure, yellowsolid (28 mg, 24%). 1H NMR (600 MHz, d6-DMSO): δ 11.960 (s, 1H, N—H),9.244 (s, 1H, pyr-2H), 8.905-8.873 (m, 2H, pyr-4,5H), 8.204-8.178 (d,J=15.6, 1H, C═CH), 8.155 (s, 1H, indole-2H), 7.987-7.961 (d, J=15.6, 1H,C═CH), 7.427-7.412 (m, 2H, indole-4,7H), 6.934-6.915 (dd, J1=9.0,J2=2.4, 1H, indole-6H), 3.853 (s, 3H, methyl). 13C NMR (150 MHz,d6-DMSO): δ 187.3, 155.1, 148.8, 147.6, 144.0, 143.7, 140.2, 134.6,132.5, 126.0, 113.4, 113.2, 112.8, 111.9, 102.9, 55.6. Melting point:176-180° C. TLC (in 4:1 ethyl acetate:hexanes) Rf=0.30. Elementalanalysis calculated C16H13N3O2: C, 68.81; H, 4.69; N, 15.05. found:68.76; H, 4.64; N, 14.99.

5-Methoxy-2-methyl-1H-indole-3-carboxaldehyde (compound 18)

To a dried two-neck 25 mL round bottom flask at 0° C., POCl3 (1.00 mL,10.8 mmol) was added to N,N-dimethylformamide (2.5 mL). After tenminutes of stirring, 2-methyl-5-methoxyindole (1.45 mg, 9.00 mmol)dissolved in DMF (5 mL) was added dropwise. After 45 minutes, 1N NaOH(50 mL) was slowly added, forming a white precipitate. The solid wasisolated by filtration, washed with cold H2O and dried under vacuum,yielding 1.52 g of white solid (90%). 1H NMR (600 MHz, d6-DMSO): δ11.875 (s, 1H, N—H), 10.009 (s, 1H, CHO), 7.570-7.566 (d, J=2.4, 1H,indole-4H), 7.282-7.268 (d, J=8.4, 1H, indole-7H), 6.797-6.778 (dd,J1=9.0, J2=2.4, 1H, indole-6H), 3.764 (s, 3H, o-methyl), 2.646 (s, 3H,c-methyl). 13C NMR (150 MHz, d6-DMSO): δ 184.1, 155.5, 148.6, 130.0,126.4, 113.7, 112.1, 111.8, 102.3, 55.2, 11.5. Melting Point: 188-192°C. TLC (ethyl acetate:hexane 4:1) Rf=0.37. Elemental analysis calculatedfor C11H11NO2: C, 69.83; H, 5.86; N, 7.40. found: C, 69.65; H, 5.95; N,7.25.

trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 19)

To a dried 250 mL two-neck round bottom flask under argon,2-methyl-5-methoxy-1H-indole-3-carboxaldehyde (1.51 g, 7.98 mmol) wasdissolved in anhydrous MeOH (30 mL). 4-Acetylpyridine (1.32 mL, 11.97mmol, 1.5 equiv.) and piperidine (0.788 mL, 7.98 mmol) were added andthe reaction was stirred under reflux. An orange solid graduallyprecipitated, and this was isolated by filtration, rinsed with chilledMeOH and dried under vacuum, yielding 2.08 g of orange solid (89%). 1HNMR (600 MHz, d6-DMSO): δ 11.909 (s, 1H, N—H), 8.812-8.802 (dd, J1=4.2,J2=1.8, 2H, pyr-2,6H), 8.097-8.072 (d, J=15.0, 1H, C═CH), 7.947-7.937(dd, J1=4.2, J2=1.8, 2H pyr-3,5H), 7.434-7.430 (d, J=2.4, 1H,indole-4H), 7.374-7.349 (d, J=15.0, 1H, C═CH), 7.315-7.301 (d, J=8.4,1H, indole-7H), 6.851-6.833 (dd, J1=8.4, J2=2.4, 1H, indole-6H), 3.860)(s, 3H, o-methyl), 2.572 (s, 3H, c-methyl). 13C NMR (150 MHz, d6-DMSO):δ 188.1, 155.2, 150.6, 145.8, 145.1, 136.6, 131.0, 126.6, 121.5, 112.8,112.3, 110.9, 109.3, 103.5, 55.6, 12.2. Melting Point: 252-256° C. TLC(ethyl acetate:hexane 4:1) Rf=0.16. Elemental analysis calculated forC18H16N2O2: C, 73.95; H, 5.52; N, 9.58. found: C, 73.76; H, 5.46; N,9.47.

trans-3-(5-Methoxy-1-methyl-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 20)

N,Ndimethylformamide (3 mL) was added to a dried 100 mL two-neck roundbottom flask under argon containing NaH (21 mg, 0.52 mmol, 60% inmineral oil, 1.2 equiv., unwashed). After stirring for five minutes, thestarting material (120 mg, 0.43 mmol) dissolved in DMF (1 mL) was addedslowly and stirred for five minutes until a homogenous red-solution wasformed. Methyl iodide (40 μL, 0.65 mmol, 1.5 equiv.) was added slowly.After two hours, sat. NH4Cl (20 mL) and H2O (20 mL) was added; this wasextracted with ethyl acetate (3×30 mL), dried with Na2SO4, filtered andconcentrated. The crude product was purified by column chromatography(methylene chloride:methanol 95:5), providing 103 mg (82%) of solid.NMR: 1H NMR (400 MHz, d6-DMSO): δ 8.821-8.806 (dd, J1=4.4, J2=1.6, 2H,pyr-2,6H), 8.143 (s, 1H, indole-2H), 8.081-8.042 (d, J=15.6, 1H, C═CH),7.950-7.935 (dd, J1=4.4, J2=1.6, 2H, pyr-3,5H), 7.517-7.473 (m, 3H,C═CH, indole-4,7H), 6.971-6.942 (dd, J1=9.2, J2=2.4, 1H, indole-6H),3.875 (s, 3H, o-methyl), 3.838 (s, 3H, n-methyl). 13C NMR (100 MHz,d6-DMSO): δ 188.2, 155.5, 150.6, 144.9, 140.2, 137.3, 133.1, 126.5,121.5, 114.2, 112.3, 111.9, 111.5, 102.9, 55.7, 33.4. Melting Point:178-182° C. TLC (in methylene chloride:methanol 95:5) Rf=0.35. Elementalanalysis calculated for C18H16N2O2.0.1H2O: C, 73.50; H, 5.55; N, 9.52.found: C, 73.34; H, 5.77; N, 9.25.

trans-3-(5-Hydroxy-1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 21)

In a dried 250 mL two-neck round bottom flask under argon, compound 19(500 mg, 1.71 mmol) was partially dissolved in anhydrous CH2Cl2 (35 mL)and placed at −78° C. BBr3 (17 mL, 1.0 M in CH2Cl2, 17 mmol) was addeddrop-wise via an addition funnel under argon. After addition, thereaction was warmed to room temperature and stirring continued for onehour. Ice-water (50 mL) was added, followed by 1N NaOH until the pH≈12(˜60 mL). The CH2Cl2 was further extracted with 1N NaOH (3×20 mL). Thebasic extracts were neutralized with 8N HCl to a neutral pH (˜8 mL),upon which the product precipitated. The precipitate was filtered anddried under vacuum, providing 454 mg (95%) of a yellow solid. NMR: 1HNMR (600 MHz, d6-DMSO): δ 11.833 (s, 1H, N—H), 9.072 (s, 1H, O—H), 8.818(s, 2H, pyr-2,6H), 8.064-8.039 (d, J=15.0, 1H, C═CH), 7.897 (s, 2H,pyr-3,5H), 7.342 (s, 1H, indole-4H), 7.282-7.257 (d, J=15.0, 1H, C═CH),7.209-7.195 (d, J=8.4, 1H, indole-7H), 6.687-6.674 (d, J=7.8, 1H,indole-6H), 2.542 (s, 3H, methyl). 13C NMR (150 MHz, d6-DMSO): δ 187.7,153.0, 150.7, 145.9, 145.4, 140.0, 130.2, 126.8, 121.3, 112.2, 112.0,111.6, 109.1, 105.2, 12.0. Melting Point: 296-299° C. TLC (in ethylacetate:hexane 4:1) Rf=0.19. Elemental analysis calculated forC17H14N2O2.0.1 CH2Cl2: C, 71.55; H, 4.99; N, 9.79. found: C, 71.89; H,5.02; N, 9.67.

2-Methyl-1H-indol-5-ol (compound 22)

A dried 250 mL two-neck round bottom flask was charged with2-methyl-5-methoxyindole (1.00 g, 6.20 mmol), purged with argon, andCH2Cl2 (30 mL) was added and stirred vigorously until the indole wasdissolved. After placing the reaction at −78° C., BBr3 (37.2 mL, 1.0 Min CH₂Cl₂, 37.2 mmol, 6 equiv.) was added dropwise via an additionfunnel under argon. After addition, the reaction was allowed to slowlywarm to room temperature. Thirty minutes after removing the coolingbath, the reaction was poured into ice-water (˜50 mL) and sat. NaHCO3(50 mL), to a neutral pH. This was extracted with CH2Cl2 (3×50 mL); theaqueous phase retained a yellow color and was acidified to pH˜3 (with 5NHCl) and extracted with ethyl acetate (2×50 mL). The combined organicextracts were washed with brine, dried with Na2SO4, filtered andconcentrated to a brown oil. After drying under vacuum for 6 hours, 845mg of a pure brown solid was isolated (93%, 147.17 MW). 1H NMR (600 MHz,d6-DMSO): 10.538 (s, 1H, N—H), 8.476 (s, 1H, O—H), 7.028-7.014 (d,J=8.4, 1H, indole-7H), 6.701-6.698 (d, J=1.8, 1H, indole-4H),6.487-6.468 (dd, J1=9.0, J2=2.4, 1H, indole-6H), 5.906 (s, 1H,indole-3H), 2.305 (s, 3H, methyl). 13C NMR (150 MHz, d6-DMSO): δ 150.4,135.8, 130.6, 129.4, 110.6, 109.8, 103.3, 98.5, 13.5. Melting point:131-134° C. TLC (in 1:1 ethyl acetate:hexanes) Rf=0.28. Elementalanalysis calculated for C9H8NO: C, 73.45; H, 6.16; N, 9.52. found: C,73.09; H, 6.29; N, 9.28.

5-(4-Methylbenzoate)methoxy-2-methyl-1H-indole (compound 23)

In a 250 mL round bottom flask, 2-methyl-1H-indole-5-ol (670 mg, 4.55mmol) was partially dissolved in CH2Cl2 (50 mL). Tetran-butylammoniumbromide (808 mg, 2.5 mmol) was added, followed by NaOH (50 mL of a 5 Nsolution, 250 mmol), and methyl 4-(bromomethyl)benzoate (1.15 g, 5.01mmol, 1.1 equiv.). After 8 hours, the organic layer was removed, and theaqueous phase was extracted with CH2Cl2 (1×30 mL). The combined extractswere washed with brine, dried with Na2SO4, filtered and concentrated toan oil. Purification by column chromatography (1:3 ethyl acetate:hexane)provided 839 mg of pure product (63%, 295.33 MW) [followed by the3,5-doubly alkylated product (115 mg, 5%)]. 1H NMR (600 MHz, d6-DMSO): δ10.755 (s, 1H, N—H), 7.980-7.966 (d, J=8.4, 2H, phenyl-3,5H),7.602-7.588 (d, J=8.4, 2H, phenyl-2,6H), 7.150-7.136 (d, J=8.4, 1H,indole-7H), 6.982-6.978 (d, J=2.4, 1H, indole-4H) 6.717-6.699 (dd,J1=8.4, J2=2.4, 1H, indole-6H), 6.005 (s, 1H, indole-3H), 5.157 (s, 2H,methylene), 3.849 (s, 3H, o-methyl), 2.331 (s, 3H, c-methyl). 13C NMR(150 MHz, d6-DMSO): δ 166.1, 151.9, 143.6, 136.3, 131.4, 129.3, 129.0,128.7, 127.4, 111.0, 110.1, 102.8, 99.0, 69.0, 52.1, 13.4. Meltingpoint: 151-154° C. TLC (in 1:1 ethyl acetate:hexanes) Rf=0.49 (doublyalkylated product Rf=0.42). Elemental analysis calculated for C19H17NO3:C, 73.20; H, 5.80; N, 4.74. found: C, 73.02; H, 5.82; N, 4.55.

5-(4-Methylbenzoate)methoxy-2-methyl-1H-indole-3-carboxaldehyde(compound 24)

To a dried two-neck 250 mL round bottom flask under argon, POCl3 (325μL, 3.5 mmol) was added to N.Ndimethylformamide (8 mL) at 0° C. Afterstirring for five minutes, compound 23 (320 mg, 1.08 mmol) dissolved inDMF (4 mL) was added dropwise. The yellow solution was slowly warmed toroom temperature and after one hour sat. NaHCO3 was added (50 mL),producing a white precipitate, followed by 1N NaOH (20 mL) to completeprecipitation (direct workup with NaOH resulted in roughly 1:1 mixtureof ester product to ester-hydrolyzed analog). The solid was filtered,rinsed with cold H2O and dried under vacuum, yielding 315 mg of whitesolid (90%). 1H NMR (600 MHz, d6-DMSO): δ 11.902 (s, 1H, N—H), 10.000(s, 1H, CHO), 7.991-7.977 (d, J=8.4, 2H, phenyl-3,5H), 7.664-7.660 (d,J=2.4, 1H, indole-4H), 7.633-7.619 (d, J=8.4, 2H, phenyl-2,6H),7.304-7.290 (d, J=8.4, 1H, indole-7H), 6.906-6.887 (dd, J1=9.0, J2=2.4,1H, indole-6H), 5.211 (s, 2H, methylene), 3.854 (s, 3H, o-methyl), 2.646(s, 3H, c-methyl). 13C NMR (150 MHz, d6-DMSO): S 184.0, 166.1, 154.3,148.7, 143.2, 130.4, 129.3, 128.8, 127.4, 126.4, 113.6, 112.3, 112.2,104.0, 69.0, 52.1, 11.5. Melting point: 224-227° C. TLC: (in ethylacetate:hexanes 4:1) Rf=0.35. Elemental analysis calculated forC19H17NO4: C, 70.58; H, 5.30; N, 4.33. found: C, 70.45; H, 5.41; N,4.51.

5-(4-Benzoate)methoxy-2-methyl-1H-indole-3-carboxaldehyde (compound 25)

To a dried two-neck 250 mL round bottom flask under argon, POCl3 (565μL, 6.1 mmol) was added to N.Ndimethylformamide (12 mL) at 0° C. Afterstirring for five minutes, compound 23 (600 mg, 2.03 mmol) dissolved inDMF (9 mL) was added dropwise. The yellow solution was slowly warmed toroom temperature. After one hour, the reaction was cooled to 0° C. and5N NaOH (90 mL) was added. After stirring for 30 minutes, 5N HCl (95 mL)was added to precipitate the product, which was filtered and driedovernight under vacuum, yielding 626 mg (99%) of white solid. 1H NMR(600 MHz, d6-DMSO): δ 11.944 (s, 1H, N—H), 10.001 (s, 1H, CHO),7.961-7.947 (d, J=8.4, 2H, phenyl-3,5H), 7.664-7.660 (d, J=2.4, 1H,indole-4H), 7.590-7.576 (d, J=8.4, 2H, phenyl-2,6H), 7.304-7.290 (d,J=8.4, 1H, indole-7H), 6.902-6.884 (dd, J1=8.4, J2=2.4, 1H, indole-6H),5.194 (s, 2H, methylene), 2.647 (s, 3H, methyl). 13C NMR (150 MHz,d6-DMSO): δ 184.0, 167.3, 154.4, 148.8, 142.5, 130.4, 129.4, 127.3,127.2, 126.4, 113.6, 112.3, 112.2, 104.0, 69.1, 11.5. Melting point:267-270° C. TLC: (in ethyl acetate:hexanes 4:1) Rf=0.25.

trans-3-[5-((4-Methylbenzoate)methoxy)-1H-Indol-3-yl)]-1-(4-pyridinyl)-2-propen-1-one(compound 26)

In a dried 100 mL two-neck round bottom flask under argon, compound 24(50 mg, 0.15 mmol) was partially dissolved in anhydrous methanol (2 mL).4-Acetyl-pyridine (26 μL, 0.23 mmol) and piperidine (7 μL, 0.075 mmol)were added and the reaction was refluxed. A yellow precipitate graduallyformed, and after 24 hours the reaction was brought to room temperatureand the solid was isolated by filtration, rinsed with cold methanol anddried under vacuum, yielding 36 mg. However, crude NMR showed 1:0.15product:aldehyde. This mixture was dry loaded onto silica and purifiedby column chromatography (ethyl acetate:hexanes 2:1), providing 22 mgpure product (34%). 1H NMR (600 MHz, d6-DMSO): δ 11.937 (s, 1H, N—H),8.829-8.819 (d, J=6.0, 2H, pyr-2,6H), 8.069-8.044 (d, J=15.0, 1H, C═CH),7.971-7.957 (d, J=8.4, 2H, phenyl-3,5H), 7.905-7.896 (d, J=5.4, 2H,pyr-3,5H), 7.662-7.649 (d, J=7.8, 2H, phenyl-2,6H), 7.479-7.475 (d,J=2.4, 1H, indole-4H), 7.326-7.312 (d, J=8.4, 1H, indole-7H),7.284-7.259 (d, J=15.0, 1H, C═CH), 6.938-6.920 (dd, J1=8.7, J2=2.1, 1H,indole-6H), 5.358 (s, 2H, methylene), 3.838 (s, 3H, o-methyl), 2.561 (s,3H, c-methyl). 13C NMR (150 MHz, d6-DMSO): δ 188.1, 166.0, 153.9, 150.6,146.0, 145.1, 143.5, 139.5, 131.3, 129.3, 128.8, 127.4, 126.4, 121.4,112.8, 112.4, 111.9, 109.3, 104.8, 69.2, 52.2, 12.1. Melting Point:236-239° C. TLC (ethyl acetate:hexanes4:1) Rf=0.26. Elemental analysiscalculated for C26H22N2O4. 0.25 C4H8O2: C, 72.31; H, 5.39; N, 6.25.found: C, 72.62; H, 5.64; N, 5.95.

trans-3-[5-((4-Carboxyphenyl)-methoxy)-1H-Indol-3-yl)]-1-(4-pyridinyl)-2-propen-1-one(compound 27)

In a dried 100 mL round bottom flask under argon, compound 25 (200 mg,0.65 mmol) was partially dissolved in anhydrous methanol (15 mL).4-Acetyl-pyridine (107 μL, 0.97 mmol, 1.5 equiv.) and piperidine (256μL, 0.65 mmol) were added and the reaction was refluxed. After 24 hours,the solid precipitate was isolated by filtration, yielding 75 mg ofproduct contaminated with aldehyde. Due to both solubility and poorfiltration, plenty of product remained in the filtrate; thus, thefiltrate was dry loaded onto silica and purified by columnchromatography (methylene chloride:methanol 9:1), yielding 85 mg ofpurified acid product (21%). 1H NMR (600 MHz, d6-DMSO): S 11.947 (s, 1H,N—H), 8.831-8.821 (dd, J1=4.2, J2=1.8, 2H, pyr-2,6H), 8.073-8.047 (d,J=15.6, 1H, C═CH), 7.954-7.941 (d, J=7.8, 2H, phenyl-3,5H), 7.904-7.894(dd, J1=4.2, J2=1.8, 2H, pyr-3,5H), 7.630-7.616 (d, J=8.4, 2H,phenyl-2,6H), 7.479-7.476 (d, J=1.8, 1H, indole-4H), 7.326-7.312 (d,J=8.4, 1H, indole-7H), 7.289-7.264 (d, J=15.0, 1H, C═CH), 6.939-6.920(dd, J1=9.0, J2=2.4, 1H, indole-6H), 5.349 (s, 2H, methylene), 2.562 (s,3H, methyl). 13C NMR (150 MHz, d6-DMSO): δ 188.1, 167.3, 154.0, 150.6,146.0, 145.1, 142.8, 139.6, 131.2, 130.3, 129.5, 127.2, 126.4, 121.4,112.8, 112.4, 111.9, 109.3, 104.8, 69.3, 12.1. Melting point: 269-273°C. TLC (methylene chloride:methanol 9:1) Rf=0.41. Elemental analysiscalculated for C25H20N2O4.0.25 CH2Cl2.0.25 CH3OH: C, 69.34; H, 4.91; N,6.34. found: 69.28; H, 5.22; N, 6.72.

2-Methyl-5-benzoyl-indole-3-carboxaldehyde and2-Methyl-6-benzoyl-indole-3-carboxaldehyde (compounds 28 and 29)

In a dried two-neck 250 mL round bottom flask under argon,N,Ndimethylformamide (339 μL, 4.4 mmol) was added to 1,2-dichloroethane(8 mL). The reaction was cooled to 0° C. and oxalyl chloride (377 μL,4.4 mmol) dissolved in 1,2-DCE (8 mL) was slowly added, forming a whiteheterogeneous mixture. The mixture was allowed to warm to roomtemperature while stirring. After fifteen minutes, the reaction wascooled to 0° C. and 2-methylindole (577 mg, 4.0 mmol) dissolved in1,2-DCE (8 mL) was slowly added, forming a dark red solution. After onehour, AICl3 (1.96 g, 14.7 mmol) was added and stirred vigorously.Benzoyl chloride (510 μL, 4.4 mmol) dissolved in 1,2-DCE (4 mL) wasslowly added and the reaction was warmed to room temperature and stirredovernight. After 24 hours, cold H2O (50 mL) was added, followed by 5NNaOH (10 mL), and the mixture was stirred. After one hour, 5N HCl (18mL) was added and this was extracted with methylene chloride (3×50 mL).The combined 1,2-DCE and methylene chloride extracts were combined,dried with Na2SO4, filtered and concentrated. The crude product mixturewas purified by column chromatography (ethyl acetate:hexanes 1:1-4:1),yielding 5-benzoyl product 28 (142 mg, 13%) and 6-benzoyl product 29(429 mg, 41%) (1:3 regioselectivity for 5 vs. 6 benzoylation).

2-Methyl-5-benzoyl-indole-3-carboxaldehyde (compound 28)

1H NMR (600 MHz, d6-DMSO): δ 12.371 (s, 1H, N—H), 10.073 (s, 1H), CHO,8.462 (s, 1H, indole-4H), 7.723-7.711 (m, 2H, phenyl-2,6H), 7.673-7.656(m, 2H, indole-6H, phenyl-4H), 7.587-7.561 (t, J=7.8, 2H, phenyl-3,5H),7.553-7.539 (d, J=8.4, 1H, indole-7H), 2.728 (s, 3H, methyl). 13C NMR(150 MHz, d6-DMSO): δ 196.1, 184.8, 150.8, 138.3, 138.0, 132.1, 130.9,129.5, 128.5, 124.9, 124.7, 123.4, 114.4, 111.6, 11.6. 1-D nOe:irradiation of peak at δ 12.371 (N—H): nOe signal enhancement seen at δ7.549 (C—H) and 2.728 (CH3). The H peak at 7.549 has ortho coupling(J=8.4), proving the benzoyl group inserted at the 5 position, not 6.Separately, irradiation of peak at δ 7.540 (C—H with ortho coupling):nOe signal enhancement seen at δ 12.370 (N—H), again proving theproximity of N—H to an ortho-coupled C7-H; signal enhancement also seenat δ 7.66 for C6-H (the benzoyl 2H triplet peak at δ 7.574 was alsoirradiated by proximity, thus signal enhancement was seen for the otherbenzoyl C—H peaks at δ 7.72 and δ 7.67). Melting point: 227-230° C. TLC(ethyl acetate:hexanes 4:1) Rf=0.32. Elemental analysis calculated forC17H13NO2_(—)0.2 C4H8O2 (trace ethyl acetate): C, 76.11: H, 5.24; N,4.99. found: C, 75.74; H, 4.94; N, 5.06.

2-Methyl-6-benzoyl-indole-3-carboxaldehyde (compound 29)

1H NMR (600 MHz, d6-DMSO): δ 12.323 (s, 1H, N—H), 10.116 (s, 1H, CHO),8.173-8.159 (d, J=8.4, 1H, indole-4H), 7.776-7.774 (d, J=1.2, 1H,indole-7H), 7.741-7.729 (m, 2H, phenyl-2,6H), 7.683-7.659 (t, J=7.2, 1H,phenyl-4H), 7.627-7.611 (dd, J1=7.8, J2=1.8, 1H, indole-5H), 7.583-7.558(t, J=7.5, 2H, phenyl-3,5H), 2.744 (s, 3H, methyl). 13C NMR (150 MHz,d6-DMSO): δ 196.3, 185.4, 152.2, 138.7, 135.3, 132.8, 131.8, 130.1,129.8, 129.1, 124.4, 120.3, 114.7, 114.4, 12.5. 1-D nOe: irradiation ofpeak at δ 12.323 (N—H): nOe signal enhancement seen at δ 7.775 (C—H withmeta coupling, thus proving benzoyl addition to position 6 of indole)and δ 2.744 (CH₃). Separately, irradiation of the peak at δ 7.775 (C—Hwith meta coupling: J=1.2) led to signal enhancement at δ 12.323 (N—Hpeak), thus proving proximity of meta-coupled C—H to N—H (the benzoyl2,6 2H peak at 7.73 was also irradiated by proximity, leading to signalenhancement of benzoyl 3,5 2H peak at δ 7.57). Melting point: 192-196°C. TLC (ethyl acetate:hexanes 4:1) Rf=0.40. Elemental analysiscalculated for C17H13NO2: C, 77.55; H, 4.98; N, 5.32. found: C, 77.28;H, 4.97; N, 5.15.

trans-3-(5-Benzoyl-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 30)

In a dried 100 mL round bottom flask under argon, compound 28 (50 mg,0.19 mmol) was dissolved in anhydrous methanol (3 mL). 4-Acetyl-pyridine(21 μL, 0.19 mmol) and piperidine (4 μL, 0.04 mmol) were added and thereaction was refluxed. After twelve hours, 0.3 equivalents of4-acetylpyridine (6.3 μL, 0.06 mmol) was added. After a total of 24hours reaction time, the reaction was cooled and the precipitate wasisolated by filtration and rinsed with cold methanol, providing 37 mg ofyellow solid. A crude 1H NMR showed a 1:3.5 ratio of product toaldehyde. The filtrate was concentrated and added to theproduct/aldehyde mixture, and 0.8 equivalents of both 4-acetyl-pyridine(17 μL, 0.15 mmol) and piperidine (15 μL, 0.15 mmol) were added to thereaction. This was refluxed for another 24 hours, after which thereaction was cooled, filtered and rinsed with cold methanol, providing 9mg (13%) pure, yellow product. 1H NMR (600 MHz, d6-DMSO): δ 12.397 (s,1H, N—H), 8.815-8.805 (m, 2H, pyr-2.6H), 8.321 (s, 1H, indole-4H),8.081-8.056 (d, J=15.0, 1H, C═CH), 7.820-7.808 (d, J=7.2, 2H,phenyl-2,6H), 7.761-7.728 (m, 4H, phenyl-4H, indole-6H, pyr-3,5H),7.644-7.619 (t, J=7.5, 2H, phenyl-3,5H), 7.587-7.573 (d, J=8.4, 1H,indole-7H), 7.347-7.321 (d, J=15.6, 1H, C═CH), 2.639 (s, 3H, methyl).13C NMR (150 MHz, d6-DMSO): δ 195.7, 187.9, 150.6, 146.9, 144.7, 138.8,138.5, 138.4, 131.9, 130.1, 129.4, 128.4, 125.2, 123.9, 123.6, 121.1,114.6, 111.9, 110.1, 12.1. Melting point: 258-262° C. TLC (ethylacetate:hexanes 4:1) Rf=0.24. Elemental analysis calculated forC24H18N2O2_(—)0.65 H2O: C, 76.24; H, 5.14; N, 7.41; C, 75.96; H, 4.75;N, 7.16.

trans-3-(6-Benzoyl-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 31)

In a dried 100 mL round bottom flask under argon, compound 29 (50 mg,0.19 mmol) was dissolved in anhydrous methanol (3 mL). 4-Acetyl-pyridine(21 μL, 0.19 mmol) and piperidine (19 μL, 0.19 mmol) were added and thereaction was refluxed. After 24 hours, the reaction was cooled, filteredand rinsed with cold hexanes and dried under vacuum, yielding 52 mg(74%) of pure yellow solid. 1H NMR (600 MHz, d6-DMSO): δ 12.350 (s, 1H,N—H), 8.832-8.222 (d, J=6.0, 2H, pyr-2,6H), 8.255-8.241 (d, J=8.4, 1H,indole-4H), 8.125-8.099 (d, J=15.6, 1H, C═CH), 8.008-7.999 (d, J=5.4,2H, pyr-3,5H), 7.801 (s, 1H, indole-7H), 7.766-7.754 (d, J=7.2, 2 H,phenyl-2,6H), 7.683-7.642 (m, 2H, phenyl-4H, indole-5H), 7.585-7.563 (m,3H, C═CH, phenyl-3,5H), 2.659 (s, 3H, methyl). 13C NMR (150 MHz,d6-DMSO): δ 195.5, 188.2, 150.6, 148.5, 144.6, 138.7, 138.2, 135.4,132.0, 130.6, 129.4, 129.2, 128.4, 123.1, 121.5, 119.9, 114.5, 114.1,109.6, 12.3. Melting point: 267-270° C. TLC (ethyl acetate:hexanes 4:1)Rf=0.29. Elemental analysis calculated for C24H18N2O2_(—)0.05 H2O: C,78.48; H, 4.97; N, 7.63. found: C, 78.08; H, 5.01; N, 7.50.

2-Methyl-5-nitro-1H-indole (compound 32)

In a 250 mL round bottom flask, 2.62 grams of 2-methyl-indole (20 mmol)was dissolved in 20 mL of H2SO4 after vigorous stirring. In a separateflask, 1.87 grams of NaNO3 (1.1×, 22 mmol, 84.99 MW) was dissolved in 20mL of H2SO4, also after vigorous stirring, and added dropwise viaaddition funnel to the indole. After addition, the reaction was stirredfor another 10 minutes, and then poured into 400 mL of ice-water,precipitating a yellow product. The product was isolated via filtrationand washed with cold water. After 12 hours of drying under vacuum, 3.35grams of yellow product was isolated (95%). 1H NMR (600 MHz, d6-DMSO): δ11.703 (s, 1H, N—H), 8.4003-8.400 (d, J=1.8, 1H, indole-4H), 7.916-7.898(dd, J1=9.0, J2=1.8, 1H, indole-6H), 7.422-7.407 (d, 1H, J=9.0, 1H,indole-7H), 6.408 (s, 1H, indole-3H), 2.419 (s, 1H, methyl). 13C NMR(150 MHz, d6-DMSO): δ 140.5, 140.0, 139.4, 128.0, 115.9, 115.7, 110.7,101.6, 13.4. 1-D nOe: irradiation of peak at δ 7.422-7.407 (C7-H): nOesignal enhancement seen at δ 11.690 (N—H) and 7.916-7.897 (C6-H).Irradiation of peak at 11.690 (N—H): nOe signal enhancement seen at δ7.422-7.407 (C7-H) and 2.419 (C2-CH3) (nOe observed between the C—Hproton with ortho coupling and N—H proton, thus proving NO2 inserted atindole C-5 vs. C-6). Melting point: 166-169° C. TLC (in 1:1 ethylacetate:hexanes) Rf=0.38. Elemental analysis calculated for C9H8N2O2: C,61.36; H, 4.58; N, 15.90. found: C, 61.54; H, 4.63; N, 15.89.

2-Methyl-5-amino-1H-indole (compound 33)

Compound 32 (1.00 grams, 5.68 mmol) was dissolved in 75 mL ethanol. 10%Pd/C was added (200 mg) and the mixture was subjected to H2 (38 psi)using a Parr hydrogenator for 3.5 hours. The mixture was filtered overcelite, which was washed with methanol. After concentration and dryingunder vacuum, 801 mg of a brown powder (97%) was isolated. 1H NMR (600MHz, d6-DMSO): δ 10.370 (s, 1H, N—H), 6.943-6.929 (d, J=8.4, 1H,indole-7H), 6.552-6.549 (d, J=1.8, 1H, indole-4H), 6.373-6.355 (dd,J1=8.4, J2=2.4, 1H), 5.815 (s, 1H, indole-6H), 4.436 (bs, 2H, NH2),2.288 (s, 3H, methyl). 13C NMR (150 MHz, d6-DMSO): δ 140.8, 135.0,129.9, 129.6, 110.5, 110.3, 102.9, 98.0, 13.5. Melting point: 147-150°C. TLC (in 3:1 ethyl acetate:hexanes) Rf=0.30; (in 1:1 ethylacetate:hexanes) Rf=0.16. Elemental analysis calculated forC9H10N2_(—)0.05 C2H6O: C, 73.61; H, 6.99; N, 18.87. found: C, 73.48; H,6.77; N, 18.81.

2-Methyl-5-azido-1H-indole (compound 34)

In an oven dried, 250 mL round bottom flask flushed with argon, compound33 (400 mg, 2.74 mmol, 146.19 MW) was dissolved in 90% AcOH (20 mL).After complete solvation, the reaction was placed at 0° C. and protectedfrom light with foil. NaNO2 (1.1×, 3.01 mmol, 208 mg, 69.00 MW)dissolved in cold H2O 2O (2 mL) was added dropwise and stirred for tenminutes. NaN3 (1.1×, 3.01 mmol, 196 mg, 65.01 MW) dissolved in cold H2O(2 mL) was added dropwise. After one hour the reaction was poured intowater (40 mL), which was extracted 3 times with CH2Cl2 (40 mL each). Theextracts were washed with sodium bicarbonate and brine (100 mL each),dried with sodium sulfate, filtered and concentrated. Purification bysilica flash chromatography (100% CH2Cl2) yielded the azide as a lightbrown solid (310 mg, 66%). 1H NMR (600 MHz, d6-DMSO): δ 11.025 (s, 1H,N—H), 7.295-7.281 (d, J=8.4, 1H, indole-7H), 7.131-7.127 (d, J=2.4, 1H,indole-4H), 6.737-6.719 (dd, J1=8.4, J2=2.4, 1H, indole-6H), 6.108-6.106(d, J=1.2, 1H, indole-3H), 2.369 (s, 1H, methyl). ¹³C NMR (150 MHz,d6-DMSO): δ 137.5, 133.9, 130.0, 129.5, 111.7, 111.6, 108.5, 99.0, 13.4.Melting point: 57-60° C. TLC (in 1:1 ethyl acetate:hexanes) Rf=0.56. IR(film): 3409 cm.-1, 2111 (aryl-azide). Elemental analysis calculated forC9H8N4: C, 62.78; H, 4.68; N, 32.54. found: C, 62.95; H, 4.68; N, 32.53.

2-Methyl-3-carboxaldehyde-5-azido-1H-indole (compound 35)

In an oven dried, 100 mL round bottom flask purged with argon, POCl3(1.5×, 3.14 mmol, 291 μL) was added to anhydrous DMF (2.0 mL) at 0° C.Compound 34 (dissolved in DMF, 2 mL) was added dropwise. The stirredreaction was slowly warmed to RT. 1N NaOH (25 mL) and water (25 mL) wasadded, forming a precipitate which was filtered and rinsed with coldwater. The resulting white solid was dried under vacuum, affording 369mg (88%). 1H NMR (600 MHz, d6-DMSO): δ 12.078 (s, 1H, N—H), 10.031 (s,1H, CHO), 7.754-7.751 (d, J=1.8, 1H, indole-4H), 7.423-7.409 (d, J=8.4,1H, indole-7H), 6.913-6.896 (dd, J1=8.4, J2=1.8, 1H, indole-6H), 2.676(s, 3H, methyl). 13C NMR (150 MHz, d6-DMSO): δ 184.3, 149.7, 133.4,133.0, 126.6, 114.4, 113.4, 112.8, 109.4, 11.5. Melting point:degradation ca. 150° C. TLC (in 1:1 ethyl acetate:hexanes) Rf=0.19.Elemental analysis calculated for C10H8N4O: C, 59.99; H, 4.03; N, 27.99.found: C, 60.03; H, 4.07; N, 27.96.

trans-3-(5-Azido-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 36)

In a dried, 100 mL round bottom flask under argon and protected fromlight, compound 35 (80 mg, 0.40 mmol) was partially dissolved inanhydrous methanol (5 mL). 4-Acetyl-pyridine (1.5 equiv., 66 μL, 0.60mmol) and piperidine (40 μL, 0.40 mmol) were added and the reaction wasstirred at 40° C. After 24 hours, the reaction was cooled to RT and theyellow precipitate was isolated via filtration and rinsed with coldmethanol. A crude 1H NMR of this product showed a ca. 1:3 ratio ofproduct to the indole starting material. This crude product wasredissolved in anhydrousmethanol (5 mL) along with the concentratedfiltrate, and another equivalent of 4-acetyl-pyridine (44 μL, 2.5 equiv.total) and five equivalents of piperidine (200 μL, 6 equiv. total) wereadded. The stirred reaction was heated at 40° C. After 12 hours, ayellow precipitate was filtered and rinsed with cold methanol and driedunder vacuum, affording 47 mg (39%) of pure product. 1H NMR (600 MHz,d6-DMSO): δ 12.105 (s, 1H, N—H), 8.816-8.806 (dd, J1=4.8, 12=1.8, 2H,pyr-2,6H), 8.066-8.041 (d, J=15, 1H, C═CH), 7.956-7.945 (dd, J1=4.8,J2=1.8, 2H, pyr-3,5H), 7.661-7.657 (d, J=2.4, 1H, indole-4H),7.455-7.441 (d, J=8.4, 1H, indole-7H), 7.401-7.375 J=15.6, 1H, C═CH),7.001-6.984 (dd, J1=8.4, J2=1.8, indole-6H), 2.587 (s, 3H, methyl). 13CNMR (150 MHz, d6-DMSO): δ 188.2, 150.6, 146.5, 144.9, 139.0, 133.9,132.9, 126.8, 121.5, 113.9, 113.8, 113.0, 110.3, 109.2, 12.2. Meltingpoint: decomposes ca. 190° C. TLC (in 4:1 ethyl acetate:hexanes)Rf=0.21. Elemental analysis calculated for C17H13N5O: C, 67.32; H, 4.32;N, 23.09. found: C, 67.37; H, 4.33; N, 23.12.

2-Methyl-5-methoxy-6-nitro-1H-indole (compound 37)

In a 100 mL round bottom flask, 2-methyl-5-methoxyindole (370 mg, 2.30mmol) was dissolved in H2SO4 (4 mL) after vigorous stirring, then placedat 0° C. In a separate flask, 1.87 grams of NaNO3 (214 mg, 2.52 mmol)was dissolved in H2SO4, (4 mL) also after vigorous stirring, chilled to0° C., and added dropwise to the indole. After addition, the reactionwas stirred for another 10 minutes, and then poured into ice-water (200mL), precipitating a yellow product, which was extracted with ethylacetate (3×100 mL). Extracts were washed with sat. NaHCO3 and brine,dried with Na2SO4, filtered and concentrated. Purification by columnchromatography (ethyl acetate:hexane 2:3-3:2) yielded 296 mg of pureproduct (62%), (followed by 73 mg of 4-nitrated product, 15%; 4:1regioselectivity). 1H NMR (600 MHz, CDCl3): δ 8.350 (s, 1H, N—H), 7.983(s, 1H, indole-7H), 7.066 (s, 1H, indole-4H), 6.211 (s, 1H, indole-3H),3.958 (s, 3H, o-methyl), 2.477 (s, 3H, c-methyl). 13C NMR (150 MHz,CDCl3): δ 148.7, 142.8, 134.7, 134.2, 129.1, 109.2, 102.7, 101.3, 57.1,14.2. Melting point: 134-138° C. TLC (in 1:1 ethyl acetate:hexanes)Rf=0.37 (4-nitro product Rf=0.25). Elemental analysis calculated forC10H10N2O3_(—)0.1 C6H14: C, 59.27; H, 5.35; N, 13.04. found: C, 59.33;H, 5.10; N, 12.98.

2-Methyl-5-methoxy-6-amino-1H-indole (compound 38)

In a hydrogenation flask, compound 37 (270 mg, 1.31 mmol) was dissolvedin 100% EtOH (35 mL) and 10% Pd/C (40 mg) was added. The mixture washydrogenated on a Parr hydrogenator at 40 p.s.i. for 45 minutes. Thered-pink solution was filtered over celite and rinsed with MeOH,concentrated and dried under vacuum, yielding 230 mg of pure product(99%). 1H NMR (400 MHz, CDCl3): δ 7.516 (s, 1H, N—H), 6.907 (s, 1H),6.607 (s, 1H), 6.052 (s, 1H, indole-3H), 3.873 (s, 3H, o-methyl), 3.733(bs, 2H, NH2), 2.365 (s, 3H, c-methyl). 13C NMR (150 MHz, d6-DMSO): δ142.8, 132.9, 131.5, 131.4, 119.5, 100.5, 98.7, 95.7, 55.6, 13.5 Meltingpoint: 144-148° C. TLC (in 3:1 ethyl acetate:hexanes) Rf=0.33. Elementalanalysis calculated for C10H10N2O3_(—)0.1 C6H14: C, 59.27; H, 5.35; N,13.04. found: C, 59.33; H, 5.10; N, 12.98.

2-Methyl-5-methoxy-6-azido-1H-indole (compound 39)

In a dried, 100 mL round bottom flask under argon, compound 38 (227 mg,1.29 mmol) was dissolved in 90% AcOH (10 mL) and placed at 0° C. Theflask was covered with foil and the reaction was conducted in low light.NaNO2 (98 mg, 1.43 mmol, 1.1 equiv) dissolved in H2O (1 mL) was addeddropwise, and the mixture stirred for 10 minutes. NaN₃ (92 mg, 1.43mmol, 1.1 equiv.) dissolved in H2O (1 mL) was added dropwise. After 45minutes, the mixture was slowly poured into H2O (25 mL) and sat. K2CO3(19 mL) to form a neutral pH. This was extracted with ethyl acetate(4×50 mL), washed with brine, dried with Na2SO4, filtered andconcentrated. The crude product was purified by column chromatography(ethyl acetate:hexanes 1:4), yielding 139 mg of pure oil (53 To). 1H NMR(600 MHz, CDCl3): δ 7.782 (s, 1H, N—H), 7.007 (s, 1H), 6.883 (s, 1H),6.145 (s, 1H, indole-3H), 3.890 (s, 3H, o-methyl), 2.388 (s, 3H,c-methyl). 13C NMR (150 MHz, CDCl3): δ 147.0, 136.1, 130.7, 126.5,123.1, 102.4, 102.0, 100.4, 56.5, 13.7. TLC (in 1:1 ethylacetate:hexanes) Rf=0.60.

2-Methyl-3-carboxaldehyde-5-methoxy-6-azido-1H-indole (compound 40)

In an oven dried, 100 mL round bottom flask, purged with argon andprotected from light with foil, POCl3 (84 μL, 0.90 mmol, 1.5 equiv.) wasadded to anhydrous DMF (1.0 mL) at 0° C. and stirred for 5 minutes.Next, compound 39 (dissolved in DMF, 1 mL) was added dropwise. Thestirred reaction was slowly warmed to RT. 1N NaOH (10 mL) and water (10mL) were added, and this was extracted with CH2Cl2 (3×20 mL). Extractswere washed with brine, dried with Na2SO4, filtered, concentrated anddried overnight under vacuum, producing 95 mg pure product (69%). 1H NMR(600 MHz, d6-DMSO): δ 11.865 (s, 1H, N—H), 10.004 (s, 1H, CHO), 7.645(s, 1H, indole-4H), 7.032 (s, 1H, indole-7H), 3.857 (s, 3H, o-methyl),2.645 (s, 3H, c-methyl). 13C NMR (150 MHz, d6-DMSO): δ 184.8, 149.5,149.4, 130.3, 124.1, 124.0, 114.4, 104.2, 103.5, 56.9, 12.2. TLC (in 1:1ethyl acetate:hexanes) Rf=0.12.

trans-3-(6-Azido-5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one(compound 41)

In a dried, 100 mL round bottom flask under argon and protected fromlight, compound 40 (70 mg, 0.30 mmol) was partially dissolved inanhydrous methanol (5 mL). 4-Acetyl-pyridine (50 μL, 0.45 mmol, 1.5equiv.) and piperidine (148 μL, 1.5 mmol, 5 equiv.) were added and thereaction was stirred at 40° C. After 7 hours, the reaction was cooled toRT and the precipitate was isolated via filtration and rinsed with coldmethanol. A crude 1H NMR of this product showed a mixture of aldehydewith trace product. This mixture was redissolved in the filtrate andmore 4-acetylpyridine (165 μL, 5 equiv.) and piperidine (148 μL, 5equiv.) was added and the mixture was set to react at 40° C. After 48hours, although starting materials are still seen on TLC, the crudereaction mixture was dry loaded onto silica and purified by columnchromatography (ethyl acetate:hexane 3:1), eluting first the aldehyde,followed by the ketone and finally the product as a yellow solid (39 mg,39%). 1H NMR (600 MHz, d6-DMSO): δ 11.784 (s, 1H, N—H), 8.815-8.805 (dd,J1=4.2, J2=1.8, 2H, pyr-2,6H), 8.073-8.047 (d, J=15.6, 1H, C═CH),7.957-7.947 (dd, J1=4.2, J2=1.8, 2H, pyr-3,5H), 7.538 (s, 1H,indole-4H), 7.410-7.384 (d, J=15.6, 1H, indole C═CH), 7.051 (s, 1H,indole-7H), 3.982 (s, 3H, o-methyl), 2.569 (s, 3H, c-methyl). 13C NMR(150 MHz, d6-DMSO): δ 188.2, 150.6, 148.5, 145.6, 145.0, 139.1, 130.6,123.6, 123.3, 121.5, 113.5, 109.5, 103.8, 103.7, 56.9, 12.3. Meltingpoint: degrades to black substance ca. 160° C. TLC (in 4:1 ethylacetate:hexanes) Rf=0.15. Elemental analysis calculated forC18H15N5O2_C4H8O2 (trace ethyl acetate): C, 64.72; H, 4.60; N, 20.74.found: C, 65.11; H, 4.71; N, 20.39.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes can be made and equivalents can be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications can be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

The publication and other material used herein to illuminate theinvention or provide additional details respecting the practice of theinvention, are incorporated by reference herein, and for convenience areprovided in the following bibliography.

Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

1. A compound having the structure of Formula I:

wherein X and Y are independently absent or halogen, oxygen, azide,nitrogen, (CO)O, O(CO), O(CO)O, (CO)N, NH(CO), NH(CO)N, NH(CO)O, orO(CO)N; wherein when X or Y is halogen or azide, then R is absent, andwherein when X or Y is nitrogen, (CO)N, O(CO)N, or NH(CO)N, then two Rgroups are present; wherein R and R₁ are independently hydrogen, alkyl,alkenyl, alkynyl, aryl, or aralkyl; wherein Ar is aryl; wherein when XRis hydrogen and Y is absent, then R is not hydrogen or methyl, andwherein when XR is halide and Y is absent, then R is not hydrogen; andpharmaceutically acceptable salts, hydrates, and optical isomersthereof.
 2. (canceled)
 3. The compound of claim 1, wherein XR is methoxybound at the 5 position, YR is methyl, and R₁ is hydrogen.
 4. A compoundof claim 3, wherein Ar is 4-pyridyl.
 5. The compound of claim 1, whichis capable of inducing methuosis. 6-10. (canceled)
 11. A method ofinducing cell death in at least one cell, comprising introducing acompound of claim 1, to at least one cell and inducing cell death.
 12. Amethod of claim 11, wherein the at least one cell is a mammalian cell.13. A method of claim 11, wherein the at least one cell isdrug-resistant.
 14. A method of claim 11, wherein the at least one cellis apoptosis-resistant.
 15. A method of claim 11, wherein the at leastone cell is a cancer cell.
 16. A method of claim 11, wherein the atleast one cell is a cancer stem cell. 17-19. (canceled)
 20. A method ofclaim 11, wherein the at least one cell is a human cell.
 21. A method ofinducing cell death in a mammal in need of such induction, comprisingadministering to a subject a pharmacologically effective amount of acompound of claim
 1. 22. The method of claim 21, wherein the mammal isselected from the group consisting of: mouse; rat; guinea pig; rabbit;cat; dog; monkey; goat; cow; horse; and human.
 23. (canceled)
 24. Amethod of ameliorating the effects of cancer in a mammal in need of suchamelioration, comprising administering to a subject a pharmacologicallyeffective amount of a compound of claim
 1. 25. A method of claim 24,wherein the cancer is selected from the group consisting of: brain,lung, pancreas, bone, bladder, colon, stomach, breast, prostate, ovary,central nervous system, or skin.
 26. The method of claim 24, wherein themammal is selected from the group consisting of: mouse; rat; guinea pig;rabbit; cat; dog; monkey; goat; cow; horse; and human.
 27. (canceled)28. The method of claim 24, wherein the method further comprisesadministering a second compound, adjuvant or additional therapeutic tothe mammal.
 29. (canceled)
 30. A composition of matter comprising acompound of claim 1 and a cancer therapeutic.
 31. A composition of claim30, wherein the cancer therapeutic is selected from the group consistingof chemotherapeutic drug; toxin; immunological response modifier;enzyme; and radioisotope.
 32. A method of treating mammalian diseasesassociated with disadvantageous proliferation of cells by administeringtherapeutic agents that cause these cells to undergo methuosis and die.33. The method of claim 32 wherein the mammalian diseases are variousforms of cancer.
 34. The method of claim 33 wherein the cancers areresistant to therapeutic agents that cause cells to die by apoptosis orother methods.
 35. (canceled)
 36. A compound of claim 1 selected fromthe group consisting of:trans-3-(2-Methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(1H-Indol-3-yl)-1-phenyl-2-propen-1-one;trans-3-(1H-Indol-3-yl)-1-(2-pyridinyl)-2-propen-1-one;trans-3-(1H-Indol-3-yl)-1-(3-pyridinyl)-2-propen-1-one;trans-3-(1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(5-Methoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(5-Phenylmethoxy-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(5-Hydroxy-1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(5-Methoxy-1H-indol-3-yl)-1-(3-pyridinyl)-2-prop en-1-one;trans-3-(5-Methoxy-1H-indol-3-yl)-1-(pyrazine)-2-propen-1-one;trans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(5-Methoxy-1-methyl-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-(5-Hydroxy-1H-Indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one;trans-3-[5-((4-Methylbenzoate)methoxy)-1H-Indol-3-yl)]-1-(4-pyridinyl)-2-propen-1-one;trans-3-[5-((4-Carboxyphenyl)-methoxy)-1H-Indol-3-yl)]-1-(4-pyridinyl)-2-propen-1-one;and pharmaceutically acceptable salts, hydrates, and optical isomersthereof. 37-40. (canceled)
 41. A compound of claim 36, wherein thecompound consists essentially oftrans-3-(5-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one.